Compounds, compositions and methods for cancer patient stratification and cancer treatment

ABSTRACT

The present invention features improved compounds, especially 
     
       
         
         
             
             
         
       
     
     methods of identifying patients having cancer using biomarkers (e.g., PDE3A, SLFN12 and/or CREB3L1) that correlate with drug sensitivity and consequently treating a stratified patient population with an agent of the invention (e.g., Compounds 1-6 disclosed herein).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional Application of U.S. Ser. No. 16/075,077, filed on Aug. 2, 2018, which is the U.S. national phase application, pursuant to 35 U.S.C. § 371, of PCT International Application Ser. No.: PCT/EP2017/052393, filed Feb. 3, 2017, designating the United States and published in English, which claims priority to and the benefit of U.S. Provisional Application No. 62/291,935, filed Feb. 5, 2016, the disclosure of each of which is incorporated herein in its entirety.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under Grant No. 3U54HG005032 awarded by the National Institutes of Health. The Government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 13, 2021, is named 167741-011214USDIV-20210413-SequenceListing.TXT and is 36,863 bytes in size.

BACKGROUND OF THE INVENTION

Cancer kills over 550,000 people in the United States and over 8 million people world-wide each year. New agents, including small molecules, molecules that impact tissue-specific growth requirements, and immunomodulatory agents, have been shown to benefit a subset of patients whose cancers have unique genomic mutations or other characteristics. Unfortunately, many cancer patients are still left without effective therapeutic options.

One approach to identify new anti-cancer agents is phenotypic screening to discover novel small molecules displaying strong selectivity between cancer cell lines, followed by predictive chemogenomics to identify the cell features associated with drug response. In the 1990s, Weinstein and colleagues demonstrated that the cytotoxic profile of a compound can be used to identify cellular characteristics, such as gene-expression profiles and DNA copy number, that correlate with drug sensitivity. The ability to identify the features of cancer cell lines that mediate their response to small molecules has strongly increased in recent years with automated high-throughput chemosensitivity testing of large panels of cell lines coupled with comprehensive genomic and phenotypic characterization of the cell lines. Phenotypic observations of small molecule sensitivity can be linked to expression patterns or somatic alterations, as in the case of trastuzumab-sensitive HER2-amplified breast cancer or erlotinib-sensitive EGFR-mutant lung cancer.

Savai et al (Expert Opinion on investigational Drugs, Vol. 19, issue 1, 2010, p. 117-131) stated that targeting cancer with phosphodiesterase inhibitors might be a promising approach for the treatment of cancer. However several phosphodiesterase inhibitors have been approved for clinical treatment, including PDE3 inhibitors milrinone, cilostazol, and levosimendan for cardiovascular indications and inhibition of platelet coagulation, as well as the PDE3 inhibitor anagrelide for thrombocythemia but for no cancer indication. The most recent quality review of PDE inhibitors (Nature Reviews Drug Discovery 13, 290-314, (2014)) barely mentions cancer. From WO 2014/164704 some new PDE3 inhibitors for the treatment of cancer are known.

Methods of characterizing malignancies at a molecular level are useful for stratifying patients, thereby quickly directing them to effective therapies. Improved methods for predicting the responsiveness of subjects having cancer are urgently required.

SUMMARY OF THE INVENTION

As described below, the present invention features methods of identifying patients having a cancer (e.g. the cancer types described herein).

that is sensitive to treatment with a phosphodiesterase 3A (PDE3A) modulator (e.g., Compounds 1-6) by detecting co-expression of PDE3A and Schlafen 12 (SLFN12) polynucleotides or polypeptides and/or a lack of decrease in expression of CREB3L1 polynucleotides or polypeptides in a cancer cell derived from such patients.

In one aspect, the invention provides a method of killing or reducing the survival of a cancer cell selected as responsive to a phosphodiesterase 3A (PDE3A) modulator involving contacting the cell with one or more PDE3A modulators compound 1, compound 2, compound 3, compound 4, compound 5, and compound 6 having the structure:

where the cell was selected as having an increase in the level of a PDE3A or Schlafen 12 (SLFN12) polypeptide or polynucleotide, or combination thereof, relative to a reference, thereby reducing the survival of the cancer cell.

In another aspect, the invention provides a method of reducing cancer cell proliferation in a subject pre-selected as having a cancer that is responsive to one or more PDE3A modulators having the structure:

comprising administering to the subject the PDE3A modulator, where the subject is pre-selected by detecting an increase in the level of a PDE3A or Schlafen 12 (SLFN12) polypeptide or polynucleotide, or combination thereof, relative to a reference, thereby reducing cancer cell proliferation in said subject.

In another aspect, the invention provides a method of identifying a subject having a cancer that is resistant to PDE3A modulation, the method comprising detecting a decrease in the level of a CREB3L1 or SLFN12 polypeptide or polynucleotide level in a biological sample of the subject relative to a reference, thereby identifying said subject as having a cancer resistant to PDE3A modulation.

In another aspect, the invention provides a method of identifying a subject having a cancer that is resistant to PDE3A modulation, the method comprising detecting a decrease in the level of a CREB3L1 polypeptide or polynucleotide level in a biological sample of the subject relative to a reference, thereby identifying said subject as having a cancer resistant to PDE3A modulation.

In another aspect, the invention provides a kit for identifying a subject having cancer that is resistant to PDE3A modulation comprising a capture reagent that binds CREB3L1 polypeptide or polynucleotide. In particular embodiments, the kit includes a capture reagent that binds SLFN12 polypeptide or polynucleotide.

In one aspect, the invention provides a compound having the structure:

or a pharmaceutically acceptable salt, or prodrug thereof.

In another aspect, the invention provides a pharmaceutical composition containing one or more pharmaceutically acceptable carriers or excipients and a compound having the structure:

or a pharmaceutically acceptable salt, or prodrug thereof.

In another aspect, the invention provides a method of treating a hyperproliferative disease, particularly cancer, comprising administering to a subject in need thereof a compound having the structure

or a pharmaceutically acceptable salt, or prodrug thereof.

In another aspect, the invention provides a method of treating a hyperproliferative disease, particularly cancer, comprising administering to a subject in need thereof a compound having the structure

or a pharmaceutically acceptable salt, or prodrug thereof, wherein said a cancer is responsive to a PDE3A modulator.

In another aspect, the invention provides a method of treating a hyperproliferative disease, particularly cancer, comprising administering to a subject in need thereof a compound having the structure

or a pharmaceutically acceptable salt, or prodrug thereof, wherein said subject has been diagnosed with a cancer responsive to a PDE3A modulator. In another aspect, the invention provides a method of treating a hyperproliferative disease, particularly cancer, comprising administering to a subject in need thereof a compound having the structure

or a pharmaceutically acceptable salt, or prodrug thereof, wherein said cancer is a bone, breast, cervical, colon, endometrium, gastrointestinal stromal tumor (GIST), head and neck, hematopoetic, kidney, leiomyosarcoma, liver, lung, lymphoid, melanoma, ovarian, pancreas, prostate, soft-tissue sarcoma, thyroid cancer, urinary tract cancer.

In another aspect, the invention provides a kit for decreasing cancer cell proliferation in a subject pre-selected as responsive to a PDE3A modulator containing a compound having the structure:

or a pharmaceutically acceptable salt, or prodrug thereof.

In another aspect, the invention provides use of a PDE3A modulator for the manufacture of a medicament for the treatment of cancer, where the PDE3A modulator is a compound having the structure:

or a pharmaceutically acceptable salt, or prodrug thereof.

In another aspect, the invention provides a PDE3A modulator for use for the treatment of cancer, where the PDE3A modulator is a compound having the structure:

or a pharmaceutically acceptable salt, or prodrug thereof.

In other embodiments, the invention provides a PDE3A modulator for use for the treatment of cancer, where the PDE3A modulator is a compound having the structure:

or a pharmaceutically acceptable salt, or prodrug thereof, whereby the cancer is bone, breast, cervical, colon, endometrium, gastrointestinal stromal tumor (GIST), head and neck, hematopoetic, kidney, leiomyosarcoma, liver, lung, lymphoid, melanoma, ovarian, pancreas, prostate, soft-tissue sarcoma, thyroid cancer, urinary tract cancer.

In various embodiments of any aspect delineated herein, the PDE3A modulator reduces an activity of PDE3A.

In various embodiments, the PDE3A modulator has the structure:

In various embodiments of any aspect delineated herein, the method involves detecting a lack of a decrease in the level of expression of CREB3L1 polypeptide or polynucleotide relative to a reference.

In various embodiments of any aspect delineated herein, the method involves detecting a decrease in the level of SLFN12.

In various embodiments of any aspect delineated herein, the biological sample is a tissue sample that includes a cancer cell.

In various embodiments, the level of the PDE3A, SLFN12, or CREB3L1 polypeptide is detected by a method selected from the group consisting of immunoblotting, mass spectrometry, and immunoprecipitation.

In various embodiments, the level of the PDE3A, SLFN12, or CREB3L1 polynucleotide is detected by a method selected from the group consisting of quantitative PCR, RNA sequencing, Northern Blot, microarray, mass spectrometry, and in situ hybridization.

In various embodiments of any aspect delineated herein, the cancer cell selected as responsive to a phosphodiesterase 3A (PDE3A) modulator expresses CREB3L1 or has no loss of CREB3L1 expression relative to a reference.

In various embodiments the cancer cell being selected as responsive to a phosphodiesterase 3A (PDE3A) modulator is a bone, breast, cervical, colon, endometrium, gastrointestinal stromal tumor (GIST), head and neck, hematopoetic, kidney, leiomyosarcoma, liver, lung, lymphoid, melanoma, ovarian, pancreas, prostate, soft-tissue sarcoma, thyroid cancer, urinary tract cancer cell.

Thus in various embodiments of any aspect delineated herein, the methods disclosed above further comprise a lack of decrease in the level of CREB3L1 polypeptide or polynucleotide relative to a reference.

In various embodiments of any aspect delineated herein, the cancer cell that is resistant to a phosphodiesterase 3A (PDE3A) modulator has decreased expression of CREB3L1 or SLFN12 or loss of CREB3L1 or SLFN12 expression relative to a reference.

In various embodiments, the cancer cell selected as responsive to a phosphodiesterase 3A (PDE3A) modulator is a melanoma, endometrium, lung, hematopoetic/lymphoid, ovarian, cervical, soft-tissue sarcoma, leiomyosarcoma, urinary tract, pancreas, thyroid, kidney, glioblastoma, or breast cancer cell. In certain embodiments, the cancer cell is not a B-cell proliferative type cancer.

In various embodiments of any aspect delineated herein, the cancer cell selected as responsive to a phosphodiesterase 3A (PDE3A) modulator has increased expression of PDE3A or Schlafen 12 (SLFN12).

In various embodiments of any aspect delineated herein, the cancer cell that is resistant to a phosphodiesterase 3A (PDE3A) modulator has decreased expression of CREB3L1 or SLFN12 or loss of CREB3L1 or SLFN12 expression relative to a Reference.

-   -   “Reference” in this context means an average expression in a         representative panel of tumor cells or tumor cell lines.

In various embodiments of any aspect delineated herein, the cancer is responsive to a PDE3A modulator.

In various embodiments, the subject has been diagnosed with a cancer responsive to a PDE3A modulator.

In various embodiments, the cancer is a melanoma, endometrium, lung, hematopoetic/lymphoid, ovarian, cervical, soft-tissue sarcoma, leiomyosarcoma, urinary tract, pancreas, thyroid, kidney, glioblastoma, or breast cancer.

In various embodiments of any aspect delineated herein, the PDE3A modulator is administered orally.

In various embodiments of any aspect delineated herein, the PDE3A modulator is administered by intravenous injection.

The invention provides methods for treating subjects having cancer identified as responsive to treatment with a PDE3A modulator selected from Compounds 1-6 by detecting co-expression of PDE3A and/or Schlafen 12 (SLFN12) polynucleotides or polypeptides and/or a lack of decrease in expression of CREB3L1 polynucleotides or polypeptides in the cancer.

Consequently the invention further provides a method of detecting expression of CREB3L1 polynucleotides or polypeptides for patient stratification using expression of CREB3L1 polynucleotides or polypeptides as a biomarker.

The invention further provides a method of detecting expression of PDE3A and/or Schlafen 12 (SLFN12) polynucleotides or polypeptides for patient stratification using expression of PDE3A and/or Schlafen 12 (SLFN12) polynucleotides or polypeptides as a biomarker.

Compositions and articles defined by the invention were isolated or otherwise manufactured in connection with the examples provided below. Other features and advantages of the invention will be apparent from the detailed description, and from the claims.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

By “Anagrelide” (IUPAC Name 6,7-dichloro-1,5-dihydroimidazo (2,1-b)quinazolin-2(3H)-one) is meant a small molecule phosphodiesterase inhibitor having the following structure:

By “Cilostamide” (IUPAC Name N-cyclohexyl-N-methyl-4-[(2-oxo-1H-quinolin-6-yl)oxy]butanamide) is meant a small molecule inhibitor having the following structure:

By “Cilostazol” (IUPAC Name 6-[4-(1-cyclohexyl-1H-tetrazol-5-yl)butoxy]-3,4-dihydro-2(1H)-quinolinone) is meant a small molecule inhibitor having the following structure:

By “DNMDP” (IUPAC Name 6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one) is meant a small molecule inhibitor having the following structure:

By “Forskolin” (IUPAC Name (3R,4aR,5S,6S,6aS,10S,10aR,10bS)-6,10,10b-Trihydroxy-3,4a,7,7,10a-pentamethyl-1-oxo-3-vinyldodecahydro-1H-benzo[N]chromen-5-ylacetate) is meant a small molecule inhibitor having the following structure:

By “Levosimendan” (IUPAC Name (E)-2-cyano-1-methyl-3-(4-(4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)phenyl)guanidine) is meant a small molecule inhibitor having the following structure:

By “Milrinone” (UPAC Name 2-methyl-6-oxo-1,6-dihydro-3,4′-bipyridine-5-carbonitrile) is meant a small molecule inhibitor having the following structure:

By “Papaverine” (IUPAC Name 1-(3,4-dimethoxybenzyl)-6,7-dimethoxyisoquinoline) is meant a small molecule inhibitor having the following structure:

By “Siguazodan” (IUPAC Name (E)-2-cyano-1-methyl-3-(4-(4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)phenyl)guanidine) is meant a small molecule inhibitor having the following structure:

By “Sildenafil” (IUPAC Name 1-[4-ethoxy-3-(6,7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-5-yl)phenylsulfonyl]-4-methylpiperazine) is meant a small molecule inhibitor having the following structure:

By “Trequinsin” (IUPAC Name 9,10-dimethoxy-3-methyl-2-(2,4,6-trimethylphenyl)imino-6,7-dihydropyrimido[6,1-a]isoquinolin-4-one) is meant a small molecule inhibitor having the following structure:

By “Vardenifil” (IUPAC Name 4-[2-ethoxy-5-(4-ethylpiperazin-1-yl)sulfonyl-phenyl]-9-methyl-7-propyl-3,5,6,8-tetrazabicyclo[4.3.0]nona-3,7,9-trien-2-one) is meant a small molecule inhibitor having the following structure:

By “Zardaverine (IUPAC Name 3-[4-(Difluoromethoxy)-3-methoxyphenyl]-1H-pyridazin-6-one)” is meant a small molecule inhibitor having the following structure:

By “Compound 1” is meant a small molecule inhibitor having the following structure:

By “Compound 2” is meant a small molecule inhibitor having the following structure:

By “Compound 3” is meant a small molecule inhibitor having the following structure:

By “Compound 4” is meant a small molecule inhibitor having the following structure:

By “Compound 5” is meant a small molecule inhibitor having the following structure:

By “Compound 6” is meant a small molecule inhibitor having the following structure:

Structures drawn include all permissible rotations about bonds.

In some embodiments, any one of the compounds Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, and Compound 6 is a small molecule phosphodiesterase inhibitor.

In some other embodiments, any one of the compounds Cilostamide, Cilostazol, DNDMP, Forskolin, Levosimendan, Milrinone, Papaverine, Siguazodan, Sildenafil, Trequinsin, Vardenifil, and Zardaverine is a small molecule phosphodiesterase inhibitor.

In some embodiments, combinations of small molecule phosphodiesterase inhibitors or modulators may be used.

In some embodiments, any combination of Compounds 1-6 may be used.

In some embodiments combinations of small molecule phosphodiesterase inhibitors or modulators, especially compounds 1-6, more particularly compound 6, together with anticancer agents may be used.

A further aspect of the invention is a method of preparing compound 6, said method comprising the step of reacting the racemate Compound 3a

with NaOCl in acetic acid at a temperature range form 10-15° C. (including 10° and 15°) to obtain the racemate Compound 6a

and subsequently performing a separation of enantiomers of Compound 6a to obtain Compound 6.

Another aspect of the invention is a method of preparing compound 6, said method further comprising optionally in a preceding step a separation of enantiomers of compound 3a

to obtain Compound 3

and the reaction step with NaOCl in acetic acid at a temperature range form 10-15° C. (including 10° and 15°) is performed with the enantiomer Compound 3 and optionally further comprising a subsequent separation of enantiomers. A further aspect of the invention is a method for the preparation of Compound 6 whereby the enantiomer Compound 3

is reacted to obtain enantiomer Compound 6

Another aspect of the invention is the use of compound 3

for the preparation of compound 6.

By “CREB3L1 polypeptide” is meant a protein or fragment thereof having at least 85% amino acid sequence identity to the sequence provided at GenBank Accession No. AAH14097.1 that is cleaved upon endoplasmic reticulum stress and has transcription factor activity. The amino acid sequence provided at GenBank Accession No. AAH14097.1 is shown below.

(SEQ ID NO.: 1) 1 mdavlepfpa drlfpgssfl dlgdlnesdf lnnahfpehl dhftenmedf sndlfssffd 61 dpvldekspl ldmeldsptp giqaehsysl sgdsapqspl vpikmedttq daehgawalg 121 hklcsimvkq eqspelpvdp laapsamaaa aamattpllg lsplsrlpip hqapgemtql 181 pvikaeplev nqflkvtped lvqmpptpps shgsdsdgsq sprslppssp vrpmarssta 241 istsplltpp hklqgtsgpl llteeekrtl iaegypiptk lpltkaeeka lkrvrrkikn 301 kisaqesrrk kkeyveclek kvetftsenn elwkkvetle nanrtllqql qklqtivtnk 361 isrpykmaat qtgtclmvaa lcfvlvlgsl vpclpefssg sqtvkedpla adgvytasqm 421 psrsllfydd gaglwedgrs tllpmeppdg weinpggpae qrprdhlqhd hldsthettk 481 ylseawpkdg gngtspdfsh skewfhdrdl gpnttikls

By “CREB3L1 polynucleotide” is meant any nucleic acid molecule, including DNA and RNA, encoding a CREB3L1 polypeptide or fragment thereof. An exemplary CREB3L1 nucleic acid sequence is provided at NCBI Ref: NM_052854.3. The sequence provided at NCBI Ref: NM_052854.3 is reproduced below:

(SEQ ID NO.: 2) 1 ccagccaggg gttcccggtt tcacagagag gaaagtgaca gaagacgtgc ggagggagac 61 gcagagacag aggagaggcc ggcagccacc cagtctcggg ggagcactta gctcccccgc 121 cccggctccc accctgtccg gggggctcct gaagccctca gccccaaccc cgggctcccc 181 atggaagcca gctgtgcccc aggaggagca ggaggaggtg gagtcggctg aatgcccacg 241 gtgcgcccgg ggcccctgag cccatcccgc tcctagccgc tgccctaagg cccccgcgcg 301 ccccgcgccc cccacccggg gccgcgccgc ctccgtccgc ccctcccccg gggcttcgcc 361 ccggacctgc cccccgcccg tttgccagcg ctcaggcagg agctctggac tgggcgcgcc 421 gccgccctgg agtgagggaa gcccagtgga agggggtccc gggagccggc tgcgatggac 481 gccgtcttgg aacccttccc ggccgacagg ctgttccccg gatccagctt cctggacttg 541 ggggatctga acgagtcgga cttcctcaac aatgcgcact ttcctgagca cctggaccac 601 tttacggaga acatggagga cttctccaat gacctgttca gcagcttctt tgatgaccct 661 gtgctggatg agaagagccc tctattggac atggaactgg actcccctac gccaggcatc 721 caggcggagc acagctactc cctgagcggc gactcagcgc cccagagccc ccttgtgccc 781 atcaagatgg aggacaccac ccaagatgca gagcatggag catgggcgct gggacacaaa 841 ctgtgctcca tcatggtgaa gcaggagcag agcccggagc tgcccgtgga ccctctggct 901 gccccctcgg ccatggctgc cgcggccgcc atggccacca ccccgctgct gggcctcagc 961 cccttgtcca ggctgcccat cccccaccag gccccgggag agatgactca gctgccagtg 1021 atcaaagcag agcctctgga ggtgaaccag ttcctcaaag tgacaccgga ggacctggtg 1081 cagatgcctc cgacgccccc cagcagccat ggcagtgaca gcgacggctc ccagagtccc 1141 cgctctctgc ccccctccag ccctgtcagg cccatggcgc gctcctccac ggccatctcc 1201 acctccccac tcctcactgc ccctcacaaa ttacagggga catcagggcc actgctcctg 1261 acagaggagg agaagcggac cctgattgct gagggctacc ccatccccac aaaactcccc 1321 ctcaccaaag ccgaggagaa ggccttgaag agagtccgga ggaaaatcaa gaacaagatc 1381 tcagcccagg agagccgtcg taagaagaag gagtatgtgg agtgtctaga aaagaaggtg 1441 gagacattta catctgagaa caatgaactg tggaagaagg tggagaccct ggagaatgcc 1501 aacaggaccc tgctccagca gctgcagaaa ctccagactc tggtcaccaa caagatctcc 1561 agaccttaca agatggccgc cacccagact gggacctgcc tcatggtggc agccttgtgc 1621 tttgttctgg tgctgggctc cctcgtgccc tgccttcccg agttctcctc cggctcccag 1681 actgtgaagg aagaccccct ggccgcagac ggcgtctaca cggccagcca gatgccctcc 1741 cgaagcctcc tattctacga tgacggggca ggcttatggg aagatggccg cagcaccctg 1801 ctgcccatgg agcccccaga tggctgggaa atcaaccccg gggggccggc agagcagcgg 1861 ccccgggacc acctgcagca tgatcacctg gacagcaccc acgagaccac caagtacctg 1921 agtgaggcct ggcctaaaga cggtggaaac ggcaccagcc ccgacttctc ccactccaag 1981 gagtggttcc acgacaggga tctgggcccc aacaccacca tcaaactctc ctaggccatg 2041 ccaagaccca ggacatagga cggacccctg gtacccagaa gaggagttct tgctcactaa 2101 cccggatccg cctcgtgccc ctgcctcctg gagcttccca ttccaggaga aaaggctcca 2161 cttcccagcc cttccttgcc cctgacattt ggactcttcc cttgggccga ccactctgtt 2221 ctcattctcc ttcccaccaa catccatccg tccttctcag acaaaccact cactgggtac 2281 cccacctcct ctctcatatg cccaacacga ccactgcctc cctgccccca cacctgcacc 2341 caaacagaca catcaacgca ccccactcac agacacccct taccccaccc ccactgtaca 2401 gagaccaaga acagaaattg tttgtaaata atgaacctta ttttttatta ttgccaatcc 2461 cctaagatat tgtattttac aaatctccct cttcccttcg cccctccctt gttttatatt 2521 ttatgaagtt agtgcgggct ttgctgctcc ctggcccagg aaagagggac tacctgaccc 2581 tcacctggca cccccctgct gctgcccaag ccgctgggcc tttttaattg ccaaactgct 2641 ctcttcatca gctcagcaca tgctttaaga aagcaaaacc aaaaaaaaaa aaaaaaagat 2701 gcagcatcaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa a

By “PDE3A polypeptide” is meant a protein or fragment thereof having at least 85% amino acid sequence identity to the sequence provided at NCBI Ref No. NP_000912.3 that catalyzes the hydrolysis of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). An exemplary human full-length PDE3A amino acid sequence is provided below:

(SEQ ID NO.: 3) MAVPGDAARVRDKPVHSGVSQAPTAGRDCHHRADPASPRDSGCRGCWGDL VLQPLRSSRKLSSALCAGSLSFLLALLVRLVRGEVGCDLEQCKEAAAAEE EEAAPGAEGGVFPGPRGGAPGGGARLSPWLQPSALLFSLLCAFFWMGLYL LRAGVRLPLAVALLAACCGGEALVQIGLGVGEDHLLSLPAAGVVLSCLAA ATWLVLRLRLGVLMIALTSAVRTVSLISLERFKVAWRPYLAYLAGVLGIL LARYVEQILPQSAEAAPREHLGSQLIAGTKEDIPVFKRRRRSSSVVSAEM SGCSSKSHRRTSLPCIPREQLMGHSEWDHKRGPRGSQSSGTSITVDIAVM GEAHGLITDLLADPSLPPNVCTSLRAVSNLLSTQLTFQAIHKPRVNPVTS LSENYTCSDSEESSEKDKLAIPKRLRRSLPPGLLRRVSSTWTTTTSATGL PTLEPAPVRRDRSTSIKLQEAPSSSPDSWNNPVMMTLTKSRSFTSSYAIS AANHVKAKKQSRPGALAKISPLSSPCSSPLQGTPASSLVSKISAVQFPES ADTTAKQSLGSHRALTYTQSAPDLSPQILTPPVICSSCGRPYSQGNPADE PLERSGVATRTPSRTDDTAQVTSDYETNNNSDSSDIVQNEDETECLREPL RKASACSTYAPETMMFLDKPILAPEPLVMDNLDSIMEQLNTWNFPIFDLV ENIGRKCGRILSQVSYRLFEDMGLFEAFKIPIREFMNYFHALEIGYRDIP YHNRIHATDVLHAVWYLTTQPIPGLSTVINDHGSTSDSDSDSGFTHGHMG YVFSKTYNVTDDKYGCLSGNIPALELMALYVAAAMHDYDHPGRTNAFLVA TSAPQAVLYNDRSVLENHHAAAAWNLFMSRPEYNFLINLDHVEFKHFRFL VIEAILATDLKKHFDFVAKFNGKVNDDVGIDWTNENDRLLVCQMCIKLAD INGPAKCKELHLQWTDGIVNEFYEQGDEEASLGLPISPFMDRSAPQLANL QESFISHIVGPLCNSYDSAGLMPGKWVEDSDESGDTDDPEEEEEEAPAPN EEETCENNESPKKKTFKRRKIYCQITQHLLQNHKMWKKVIEEEQRLAGIE NQSLDQTPQSHSSEQIQAIKEEEEEKGKPRGEEIPTQKPDQ Three PDE3A isoforms are known: PDE3A1, PDE3A2, and PDE3A3. PDE3A1 comprises amino acids 146-1141, PDE3A2 isoform 2 comprises amino acids 299-1141, and PDE3A3 comprises amino acids 483-1141 of the full-length PDE3A amino acid sequence.

By “PDE3A polynucleotide” is meant any nucleic acid molecule, including DNA and RNA, encoding a PDE3A polypeptide or fragment thereof. An exemplary PDE3A nucleic acid sequence is provided at NCBI Ref: NM_000921.4:

(SEQ ID NO.: 4) 1 gggggccact gggaattcag tgaagagggc accctatacc atggcagtgc ccggcgacgc 61 tgcacgagtc agggacaagc ccgtccacag tggggtgagt caagccccca cggcgggccg 121 ggactgccac catcgtgcgg accccgcatc gccgcgggac tcgggctgcc gtggctgctg 181 gggagacctg gtgctgcagc cgctccggag ctctcggaaa ctttcctccg cgctgtgcgc 241 gggctccctg tcctttctgc tggcgctgct ggtgaggctg gtccgcgggg aggtcggctg 301 tgacctggag cagtgtaagg aggcggcggc ggcggaggag gaggaagcag ccccgggagc 361 agaagggggc gtcttcccgg ggcctcgggg aggtgctccc gggggcggtg cgcggctcag 421 cccctggctg cagccctcgg cgctgctctt cagtctcctg tgtgccttct tctggatggg 481 cttgtacctc ctgcgcgccg gggtgcgcct gcctctggct gtcgcgctgc tggccgcctg 541 ctgcgggggg gaagcgctcg tccagattgg gctgggcgtc ggggaggatc acttactctc 601 actccccgcc gcgggggtgg tgctcagctg cttggccgcc gcgacatggc tggtgctgag 661 gctgaggctg ggcgtcctca tgatcgcctt gactagcgcg gtcaggaccg tgtccctcat 721 ttccttagag aggttcaagg tcgcctggag accttacctg gcgtacctgg ccggcgtgct 781 ggggatcctc ttggccaggt acgtggaaca aatcttgccg cagtccgcgg aggcggctcc 841 aagggagcat ttggggtccc agctgattgc tgggaccaag gaagatatcc cggtgtttaa 901 gaggaggagg cggtccagct ccgtcgtgtc cgccgagatg tccggctgca gcagcaagtc 961 ccatcggagg acctccctgc cctgtatacc gagggaacag ctcatggggc attcagaatg 1021 ggaccacaaa cgagggccaa gaggatcaca gtcttcagga accagtatta ctgtggacat 1081 cgccgtcatg ggcgaggccc acggcctcat taccgacctc ctggcagacc cttctcttcc 1141 accaaacgtg tgcacatcct tgagagccgt gagcaacttg ctcagcacac agctcacctt 1201 ccaggccatt cacaagccca gagtgaatcc cgtcacttcg ctcagtgaaa actatacctg 1261 ttctgactct gaagagagct ctgaaaaaga caagcttgct attccaaagc gcctgagaag 1321 gagtttgcct cctggcttgt tgagacgagt ttcttccact tggaccacca ccacctcggc 1381 cacaggtcta cccaccttgg agcctgcacc agtacggaga gaccgcagca ccagcatcaa 1441 actgcaggaa gcaccttcat ccagtcctga ttcttggaat aatccagtga tgatgaccct 1501 caccaaaagc agatccttta cttcatccta tgctatttct gcagctaacc atgtaaaggc 1561 taaaaagcaa agtcgaccag gtgccctcgc taaaatttca cctctttcat cgccctgctc 1621 ctcacctctc caagggactc ctgccagcag cctggtcagc aaaatttctg cagtgcagtt 1681 tccagaatct gctgacacaa ctgccaaaca aagcctaggt tctcacaggg ccttaactta 1741 cactcagagt gccccagacc tatcccctca aatcctgact ccacctgtta tatgtagcag 1801 ctgtggcaga ccatattccc aagggaatcc tgctgatgag cccctggaga gaagtggggt 1861 agccactcgg acaccaagta gaacagatga cactgctcaa gttacctctg attatgaaac 1921 caataacaac agtgacagca gtgacattgt acagaatgaa gatgaaacag agtgcctgag 1981 agagcctctg aggaaagcat cggcttgcag cacctatgct cctgagacca tgatgtttct 2041 ggacaaacca attcttgctc ccgaacctct tgtcatggat aacctggact caattatgga 2101 gcagctaaat acttggaatt ttccaatttt tgatttagtg gaaaatatag gaagaaaatg 2161 tggccgtatt cttagtcagg tatcttacag actttttgaa gacatgggcc tctttgaagc 2221 ttttaaaatt ccaattaggg aatttatgaa ttattttcat gctttggaga ttggatatag 2281 ggatattcct tatcataaca gaatccatgc cactgatgtt ttacatgctg tttggtatct 2341 tactacacag cctattccag gcctctcaac tgtgattaat gatcatggtt caaccagtga 2401 ttcagattct gacagtggat ttacacatgg acatatggga tatgtattct caaaaacgta 2461 taatgtgaca gatgataaat acggatgtct gtctgggaat atccctgcct tggagttgat 2521 ggcgctgtat gtggctgcag ccatgcacga ttatgatcat ccaggaagga ctaatgcttt 2581 cctggttgca actagtgctc ctcaggcggt gctatataac gatcgttcag ttttggagaa 2641 tcatcacgca gctgctgcat ggaatctttt catgtcccgg ccagagtata acttcttaat 2701 taaccttgac catgtggaat ttaagcattt ccgtttcctt gtcattgaag caattttggc 2761 cactgacctg aagaaacact ttgacttcgt agccaaattt aatggcaagg taaatgatga 2821 tgttggaata gattggacca atgaaaatga tcgtctactg gtttgtcaaa tgtgtataaa 2881 gttggctgat atcaatggtc cagctaaatg taaagaactc catcttcagt ggacagatgg 2941 tattgtcaat gaattttatg aacagggtga tgaagaggcc agccttggat tacccataag 3001 ccccttcatg gatcgttctg ctcctcagct ggccaacctt caggaatcct tcatctctca 3061 cattgtgggg cctctgtgca actcctatga ttcagcagga ctaatgcctg gaaaatgggt 3121 ggaagacagc gatgagtcag gagatactga tgacccagaa gaagaggagg aagaagcacc 3181 agcaccaaat gaagaggaaa cctgtgaaaa taatgaatct ccaaaaaaga agactttcaa 3241 aaggagaaaa atctactgcc aaataactca gcacctctta cagaaccaca agatgtggaa 3301 gaaagtcatt gaagaggagc aacggttggc aggcatagaa aatcaatccc tggaccagac 3361 ccctcagtcg cactcttcag aacagatcca ggctatcaag gaagaagaag aagagaaagg 3421 gaaaccaaga ggcgaggaga taccaaccca aaagccagac cagtgacaat ggatagaatg 3481 ggctgtgttt ccaaacagat tgacttgtca aagactctct tcaagccagc acaacattta 3541 gacacaacac tgtagaaatt tgagatgggc aaatggctat tgcattttgg gattcttcgc 3601 attttgtgtg tatattttta cagtgaggta cattgttaaa aactttttgc tcaaagaagc 3661 tttcacattg caacaccagc ttctaaggat tttttaagga gggaatatat atgtgtgtgt 3721 gtatataagc tcccacatag atacatgtaa aacatattca cacccatgca cgcacacaca 3781 tacacactga aggccacgat tgctggctcc acaatttagt aacatttata ttaagatata 3841 tatatagtgg tcactgtgat ataataaatc ataaaggaaa ccaaatcaca aaggagatgg 3901 tgtggcttag caaggaaaca gtgcaggaaa tgtaggttac caactaagca gcttttgctc 3961 ttagtactga gggatgaaag ttccagagca ttatttgaat tctgatacat cctgccaaca 4021 ctgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgaaaga gagacagaag 4081 ggaatggttt gagagggtgc ttgtgtgcat gtgtgtgcat atgtaaagag atttttgtgg 4141 tttaagtaac tcagaatagc tgtagcaaat gactgaatac atgtgaacaa acagaaggaa 4201 gttcactctg gagtgtcttt gggaggcagc cattccaaat gccctcctcc atttagcttc 4261 aataaagggc cttttgctga tggagggcac tcaagggctg ggtgagaggg ccacgtgttt 4321 ggtattacat tactgctatg caccacttga aggagctcta tcaccagcct caaacccgaa 4381 agactgaggc attttccagt ctacttgcct aatgaatgta taggaactgt ctatgagtat 4441 ggatgtcact caactaagat caaatcacca tttaagggga tggcattctt tatacctaaa 4501 cacctaagag ctgaagtcag gtcttttaat caggttagaa ttctaaatga tgccagagaa 4561 ggcttgggaa attgtacttc agcgtgatag cctgtgtctt cttaatttgc tgcaaaatat 4621 gtggtagaga aagaaaagga aacagaaaaa tcactctggg ttatatagca agagatgaag 4681 gagaatattt caacacaggg tttttgtgtt gacataggaa aagcctgatt cttggcaact 4741 gttgtagttt gtctttcagg ggtgaaggtc ccactgacaa cccctgttgt ggtgttccac 4801 acgctgtttg ttggggtagc ttccatcggc agtctggccc attgtcagtc atgcttcttc 4861 tggccgggga gattatagag agattgtttg aagattgggt tattattgaa agtctttttt 4921 tttgtttgtt ttgttttggt ttgtttgttt atctacactt gtttatgctg tgagccaaac 4981 ctctatttaa aaagttgata ctcactttca atattttatt tcatattatt atatatgtca 5041 tgatagttat cttgatgtaa atatgaagat ttttttgttt ctgtagatag taaactcttt 5101 ttttaaaaaa ggaaaaggga aacattttta taaagttata ttttaatcac catttttata 5161 cattgtagtt ctctccaagc ccagtaagag aatgatgatt catttgcatg gaggtcgatg 5221 gacaaccaat catctacctt ttctaattta aatgataatc tgatatagtt ttattgccag 5281 ttaaatgagg atgctgcaaa gcatgttttt tcactagtaa cttttgctaa ctgaatgaat 5341 tctgggtcca tatctcccag atgaaaaact gttaaccaat accatatttt atagttggtg 5401 tccatttctt tccaacactg tttgttatga ttcttccttg agtacttata tacagacctg 5461 ctcattatct aaacaatctt accttctaag taaaccttga ttgtgatttc cagtttttat 5521 tttctctgac gtagtagaaa ggaatgttta cattaaaaat acttttgttt ctcataaatg 5581 gatattgtac tccccccttt caaagcatta ttttacaata attcatggca ttttaaaaaa 5641 taaggcaaag ataatacgac aaaaaatata catggtttca aggcaaattc tccaataagt 5701 tggaaaatgt aaaaaggatc aagtggatgc agcctctacc taaataatta aaatatattt 5761 cagtatattt ctgaattaac accaggtctt cattatttag aacttactaa attgttttca 5821 ttttcttagt tttacctgtg tatctccatg tttgcaaaaa ttactataag tcaaattttg 5881 ccagtgaatt taactatttt tctttccttg caattaaggg gaaaaaagca tttatcttat 5941 cttctcatac cccttgcatc taagtactta gcaaagtcaa tattttccca ttttccaaat 6001 gcgtccatct ctaacataaa tattaattga acatagagct atgtttggag tgagtggact 6061 ggcaggacag ttggaagtcc atcacagtct attgacagtt tcatcaaagc tgtatagtcc 6121 aactagtggg gcagcttggc tactatggtg gaagtctcag caaactgcct ggttttgttt 6181 gtttgttttg ttttaaggta caggaaataa gaggaataat agtggccaaa gcaattagaa 6241 catcttcatt ccagaactgt gttcagcaat ccaggcagat tgatacattt ttctttaaaa 6301 ataaattgct attacagcta gacgtcaatt gggataaata aagggatgaa gatccactaa 6361 gtttgtgact ttcatacaca cccagtacat ctcaaaggat gctaagggac attttctgcc 6421 agtagagttc tccccctttt tggtgacagc aatattatta tgttcacatc taactccaga 6481 gcttacttcc tgtggtgcca atgtatttgt tgcaatttac tacattttta tatgagccta 6541 tttataggtg ccattaaact caggtctttc aaatgaaaga gtttctagcc cacttaggga 6601 aaaagataat tgtttagaaa accataaaat caatggtagg aaaagttgga actggttacc 6661 tggatgccat ggttctctgt taaataaagt aagagaccag gtgtattctg agtgtcatca 6721 gtgttatttt cagcatgcta ataaatgtct ttccggttat atatctatct aaattaacct 6781 ttaaaatatt ggtttccttg ataaaagcac cacttttgct tttgttagct gtaatatttt 6841 ttgtcattta gataagacct ggtttggctc tcaataaaag atgaagacag tagctctgta 6901 cagggatata tctatattag tcttcatctg atgaatgaag aaattttctc atattatgtt 6961 caagaaagta tttacttcct aaaaatagaa ttcccgattc tgtctatttt ggttgaatac 7021 cagaacaaat ctttccgttg caatcccagt aaaacgaaag aaaaggaata tcttacagac 7081 tgttcatatt agatgtatgt agactgttaa tttgcaattt ccccatattt cctgcctatc 7141 ttacccagat aactttcttt gaaggtaaaa gctgtgcaaa aggcatgaga ctcaggccta 7201 ctctttgttt aaatgatgga aaaatataaa ttattttcta agtaataaaa gtataaaaat 7261 tatcattata aataaagtct aaagtttgaa attattaatt taaaaaaaaa aaaaaaaaa

By “Schlafen 12 (SLFN12) polypeptide” is meant a protein or fragment thereof having at least 85% amino acid sequence identity to the sequence provided at NCBI Ref No. NP_060512.3 that interacts with PDE3A when bound to one of the compounds described herein. An exemplary human SLFN12 amino acid sequence is provided below:

(SEQ ID NO.: 5) MNISVDLETNYAELVLDVGRVTLGENSRKKMKDCKLRKKQNESVSRAMCA LLNSGGGVIKAEIENEDYSYTKDGIGLDLENSFSNILLFVPEYLDFMQNG NYFLIFVKSWSLNTSGLRITTLSSNLYKRDITSAKVMNATAALEFLKDMK KTRGRLYLRPELLAKRPCVDIQEENNMKALAGVFFDRTELDRKEKLTFTE STHVEIKNFSTEKLLQRIKEILPQYVSAFANTDGGYLFIGLNEDKEIIGF KAEMSDLDDLEREIEKSIRKMPVHHFCMEKKKINYSCKFLGVYDKGSLCG YVCALRVERFCCAVFAKEPDSWHVKDNRVMQLTRKEWIQFMVEAEPKFSS SYEEVISQINTSLPAPHSWPLLEWQRQRHHCPGLSGRITYTPENLCRKLF LQHEGLKQLICEEMDSVRKGSLIFSRSWSVDLGLQENHKVLCDALLISQD SPPVLYTFHMVQDEEFKGYSTQTALTLKQKLAKIGGYTKKVCVMTKIFYL SPEGMTSCQYDLRSQVIYPESYYFTRRKYLLKALFKALKRLKSLRDQFSF AENLYQIIGIDCFQKNDKKMFKSCRRLT

By “Schlafen 12 (SLFN12) polynucleotide” is meant any nucleic acid molecule, including DNA and RNA, encoding a SLFN12 polypeptide or fragment thereof. An exemplary SLFN12 nucleic acid sequence is provided at NCBI Ref: NM_018042.4:

(SEQ ID NO.: 6) 1 tttgtaactt cacttcagcc tcccattgat cgctttctgc aaccattcag actgatctcg 61 ggctcctatt tcatttacat tgtgtgcaca ccaagtaacc agtgggaaaa ctttagaggg 121 tacttaaacc ccagaaaatt ctgaaaccgg gctcttgagc cgctatcctc gggcctgctc 181 ccaccctgtg gagtgcactt tcgttttcaa taaatctctg cttttgttgc ttcattcttt 241 ccttgctttg tttgtgtgtt tgtccagttc tttgttcaac acgccaagaa cctggacact 301 cttcactggt aacatatttt ggcaagccaa ccaggagaaa agaatttctg cttggacact 361 gcatagctgc tgggaaaatg aacatcagtg ttgatttgga aacgaattat gccgagttgg 421 ttctagatgt gggaagagtc actcttggag agaacagtag gaaaaaaatg aaggattgta 481 aactgagaaa aaagcagaat gaaagtgtct cacgagctat gtgtgctctg ctcaattctg 541 gagggggagt gatcaaggct gaaattgaga atgaagacta tagttataca aaagatggaa 601 taggactaga tttggaaaat tcttttagta acattctgtt atttgttcct gagtacttag 661 acttcatgca gaatggtaac tactttctga tttttgtgaa gtcatggagc ttgaacacct 721 ctggtctgcg gattaccacc ttgagctcca atttgtacaa aagagatata acatctgcaa 781 aagtcatgaa tgccactgct gcactggagt tcctcaaaga catgaaaaag actagaggga 841 gattgtattt aagaccagaa ttgctggcaa agaggccctg tgttgatata caagaagaaa 901 ataacatgaa ggccttggcc ggggtttttt ttgatagaac agaacttgat cggaaagaaa 961 aattgacctt tactgaatcc acacatgttg aaattaaaaa cttctcgaca gaaaagttgt 1021 tacaacgaat taaagagatt ctccctcaat atgtttctgc atttgcaaat actgatggag 1081 gatatttgtt cattggttta aatgaagata aagaaataat tggctttaaa gcagagatga 1141 gtgacctcga tgacttagaa agagaaatcg aaaagtccat taggaagatg cctgtgcatc 1201 acttctgtat ggagaagaag aagataaatt attcatgcaa attccttgga gtatatgata 1261 aaggaagtct ttgtggatat gtctgtgcac tcagagtgga gcgcttctgc tgtgcagtgt 1321 ttgctaaaga gcctgattcc tggcatgtga aagataaccg tgtgatgcag ttgaccagga 1381 aggaatggat ccagttcatg gtggaggctg aaccaaaatt ttccagttca tatgaagagg 1441 tgatctctca aataaatacg tcattacctg ctccccacag ttggcctctt ttggaatggc 1501 aacggcagag acatcactgt ccagggctat caggaaggat aacgtatact ccagaaaacc 1561 tttgcagaaa actgttctta caacatgaag gacttaagca attaatatgt gaagaaatgg 1621 actctgtcag aaagggctca ctgatcttct ctaggagctg gtctgtggat ctgggcttgc 1681 aagagaacca caaagtcctc tgtgatgctc ttctgatttc ccaggacagt cctccagtcc 1741 tatacacctt ccacatggta caggatgagg agtttaaagg ctattctaca caaactgccc 1801 taaccttaaa gcagaagctg gcaaaaattg gtggttacac taaaaaagtg tgtgtcatga 1861 caaagatctt ctacttgagc cctgaaggca tgacaagctg ccagtatgat ttaaggtcgc 1921 aagtaattta ccctgaatcc tactatttta caagaaggaa atacttgctg aaagcccttt 1981 ttaaagcctt aaagagactc aagtctctga gagaccagtt ttcctttgca gaaaatctat 2041 accagataat cggtatagat tgctttcaga agaatgataa aaagatgttt aaatcttgtc 2101 gaaggctcac ctgatggaaa atggactggg ctactgagat atttttcatt atatatttga 2161 taacattctc taattctgtg aaaatatttc tttgaaaact ttgcaagtta agcaacttaa 2221 tgtgatgttg gataattggg ttttgtctat tttcacttct ccctaaataa tcttcacaga 2281 tattgtttga gggatattag gaaaattaat ttgttaactc gtctgtgcac agtattattt 2341 actctgtctg tagttcctga ataaattttc ttccatgctt gaactgggaa aattgcaaca 2401 cttttattct taatgacaac agtgaaaatc tcccagcata tacctagaaa acaattataa 2461 cttacaaaag attatccttg atgaaactca gaatttccac agtgggaatg aataagaagg 2521 caaaactcat

In some aspects, the compound is an isomer. “Isomers” are different compounds that have the same molecular formula. “Stereoisomers” are isomers that differ only in the way the atoms are arranged in space. As used herein, the term “isomer” includes any and all geometric isomers and stereoisomers. For example, “isomers” include geometric double bond cis- and trans-isomers, also termed E- and Z-isomers; R- and S-enantiomers; diastereomers, (d)-isomers and (l)-isomers, racemic mixtures thereof; and other mixtures thereof, as falling within the scope of this invention

The symbol

denotes a bond that can be a single, double or triple bond as described herein. Provided herein are various geometric isomers and mixtures thereof resulting from the arrangement of substituents around a carbon-carbon double bond or arrangement of substituents around a carbocyclic ring. Substituents around a carbon-carbon double bond are designated as being in the “Z” or “E” configuration wherein the terms “Z” and “E” are used in accordance with IUPAC standards. Unless otherwise specified, structures depicting double bonds encompass both the “E” and “Z” isomers.

Substituents around a carbon-carbon double bond alternatively can be referred to as “cis” or “trans,” where “cis” represents substituents on the same side of the double bond and “trans” represents substituents on opposite sides of the double bond. The arrangement of substituents around a carbocyclic ring can also be designated as “cis” or “trans.” The term “cis” represents substituents on the same side of the plane of the ring, and the term “trans” represents substituents on opposite sides of the plane of the ring. Mixtures of compounds wherein the substituents are disposed on both the same and opposite sides of plane of the ring are designated “cis/trans.”

The term “enantiomers” refers to a pair of stereoisomers that are non-superimposable mirror images of each other. An atom having an asymmetric set of substituents can give rise to an enantiomer. A mixture of a pair of enantiomers in any proportion can be known as a “racemic” mixture. The term “(±)” is used to designate a racemic mixture where appropriate. “Diastereoisomers” are stereoisomers that have at least two asymmetric atoms, but which are not mirror-images of each other. The absolute stereochemistry is specified according to the Cahn-Ingold-Prelog R-S system. When a compound is an enantiomer, the stereochemistry at each chiral carbon can be specified by either R or S. Resolved compounds whose absolute configuration is unknown can be designated (+) or (−) depending on the direction (dextro- or levorotatory) which they rotate plane polarized light at the wavelength of the sodium D line. Certain of the compounds described herein contain one or more asymmetric centers and can thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that can be defined, in terms of absolute stereochemistry at each asymmetric atom, as (R)- or (S)-. The present chemical entities, pharmaceutical compositions and methods are meant to include all such possible isomers, including racemic mixtures, optically substantially pure forms and intermediate mixtures.

Optically active (R)- and (S)-isomers can be prepared, for example, using chiral synthons or chiral reagents, or resolved using conventional techniques. Enantiomers can be isolated from racemic mixtures by any method known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC), the formation and crystallization of chiral salts, or prepared by asymmetric syntheses.

Optical isomers can be obtained by resolution of the racemic mixtures according to conventional processes, e.g., by formation of diastereoisomeric salts, by treatment with an optically active acid or base. Examples of appropriate acids are tartaric, diacetyltartaric, dibenzoyltartaric, ditoluoyltartaric, and camphorsulfonic acid. The separation of the mixture of diastereoisomers by crystallization followed by liberation of the optically active bases from these salts affords separation of the isomers. Another method involves synthesis of covalent diastereoisomeric molecules by reacting disclosed compounds with an optically pure acid in an activated form or an optically pure isocyanate. The synthesized diastereoisomers can be separated by conventional means such as chromatography, distillation, crystallization or sublimation, and then hydrolyzed to deliver the enantiomerically enriched compound. Optically active compounds can also be obtained by using active starting materials. In some embodiments, these isomers can be in the form of a free acid, a free base, an ester or a salt.

In certain embodiments, the compound of the invention can be a tautomer. As used herein, the term “tautomer” is a type of isomer that includes two or more interconvertible compounds resulting from at least one formal migration of a hydrogen atom and at least one change in valency (e.g., a single bond to a double bond, a triple bond to a single bond, or vice versa). “Tautomerization” includes prototropic or proton-shift tautomerization, which is considered a subset of acid-base chemistry. “Prototropic tautomerization” or “proton-shift tautomerization” involves the migration of a proton accompanied by changes in bond order. The exact ratio of the tautomers depends on several factors, including temperature, solvent, and pH. Where tautomerization is possible (e.g., in solution), a chemical equilibrium of tautomers can be reached. Tautomerizations (i.e., the reaction providing a tautomeric pair) can be catalyzed by acid or base, or can occur without the action or presence of an external agent. Exemplary tautomerizations include, but are not limited to, keto-to-enol; amide-to-imide; lactam-to-lactim; enamine-to-imine; and enamine-to-(a different) enamine tautomerizations. A specific example of keto-enol tautomerization is the interconversion of pentane-2,4-dione and 4-hydroxypent-3-en-2-one tautomers. Another example of tautomerization is phenol-keto tautomerization. A specific example of phenol-keto tautomerization is the interconversion of pyridin-4-ol and pyridin-4(1H)-one tautomers.

All chiral, diastereomeric, racemic, and geometric isomeric forms of a structure are intended, unless specific stereochemistry or isomeric form is specifically indicated. All processes used to prepare compounds of the present invention and intermediates made therein are considered to be part of the present invention. All tautomers of shown or described compounds are also considered to be part of the present invention.

By “agent” is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.

By “ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.

By “alteration” is meant a change (increase or decrease) in the expression levels or activity of a gene or polypeptide as detected by standard art known methods such as those described herein. As used herein, in one embodiment an alteration includes an about 10% change in expression levels, preferably an about 25% change, more preferably an about 40% change, and most preferably an about 50% or greater change in expression levels. In certain embodiments an alteration includes a 10% or less (including 10%) change in expression levels, preferably a 25% or less (including 25%) change, more preferably a40% or less (including 40%) change, and most preferably a 50% or less (including 50%) or greater change in expression levels. In other embodiments an alteration includes a 9%-11% (including 9% and 11%) change in expression levels, preferably a 10%-25% (including 10% and 25%) change, more preferably a 25%-40% (including 25% and 40%) change, and most preferably a 40%-50% (including 40%-50%) or greater than 50% (including 50%) change in expression levels. In other certain embodiments an alteration includes a 9%-11% (including 9% and 11%) change in expression levels, preferably a 22%-28% (including 22% and 28%) change, more preferably a 35%-45% (including 35% and 45%) change, and most preferably a 45%-55% (including 45%-55%) or a greater or equal to 55% change in expression levels

By “analog” is meant a molecule that is not identical, but has analogous functional or structural features. For example, a polypeptide analog retains the biological activity of a corresponding naturally-occurring polypeptide, while having certain biochemical modifications that enhance the analog's function relative to a naturally occurring polypeptide. Such biochemical modifications could increase the analog's protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding. An analog may include an unnatural amino acid.

In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. Patent law and can mean “includes,” “including,” and the like; “consisting essentially of” or “consists essentially” likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

“Detect” refers to identifying the presence, absence or amount of the analyte to be detected.

In particular embodiments, the analyte is a PDE3A or SLFN12 polypeptide.

By “disease” is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include melanoma, adenocarcinoma, lung cancer, cervical cancer, liver cancer and breast cancer.

By “effective amount” is meant the amount of a compound described herein required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject.

Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an “effective” amountIn still other embodiments, the PDE3A modulator is Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, or Compound 6.

The invention provides a number of targets that are useful for the development of highly specific drugs to treat or a disorder characterized by the methods delineated herein. In addition, the methods of the invention provide a facile means to identify therapies that are safe for use in subjects.

In addition, the methods of the invention provide a route for analyzing virtually any number of compounds for effects on a disease described herein with high-volume throughput, high sensitivity, and low complexity.

By “fragment” is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% of the entire length of the reference nucleic acid molecule or polypeptide. In certain embodiments this portion contains, preferably, at least 9%-11% (including 9% and 11%), 18%-22% (including 18% ands 22%), 27%-33% (including 27% and 33%), 36%-44% (including 36% and 44%), 45%-55% (including 45% and 55%), 54%-66% (including 54% and 66%), 63%-77% (including 63% and 77%), 72%-88% (including 72% and 88%), or 81%-99% (including 81% and 99%) of the entire length of the reference nucleic acid molecule or polypeptide A fragment may contain about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, or about 1000 nucleotides or amino acids. In certain embodiments a fragment may contain 9-11, about 18-22, 27-33, 36-44, 45-55, 54-66, 63-77, 72-88, 81-99, 90-110, 180-220, 270-330, 360-440, 450-550, 540-660, 630-770, 720-880, 810-990, or 900-1100 nucleotides or amino acids (including for each the mentioned limitation e.g. for “9-11” means including 9 and 11.

“Hematological tumors” include aggressive and indolent forms of leukemia and lymphoma, namely non-Hodgkins disease, chronic and acute myeloid leukemia (CML/AML), acute lymphoblastic leukemia (ALL), Hodgkins disease, multiple myeloma and T-cell lymphoma. Also included are myelodysplastic syndrome, plasma cell neoplasia, paraneoplastic syndromes, and cancers of unknown primary site as well as AIDS related malignancies.

“Hybridization” means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.

“Hyperproliferative disease” includes for example psoriasis, keloids and other hyperplasias which affect the skin, benign hyperproliferative diseases, hematopoietic hyperproliferative diseases, cancer (especially metastatic or malignant tumors, more specifically solid tumors and haematological tumors).

“Benign hyperproliferative diseases” include for example, endometriosis, leiomyoma and benign prostate hyperplasia.

“Hematopoietic hyperproliferative diseases” also known as myoproliferative disorders include e.g. polycythemia vera, essential thrombocytosis, thrombocytosis, primary myelofibrosis, and others.

By “marker” or “biomarker” is meant any protein or polynucleotide having an alteration in expression level or activity (e.g., at the protein or mRNA level) that is associated with a disease or disorder. In particular embodiments, a marker of the invention is PDE3A or SLFN12 or CREB3L1.

By “modulator” is meant any agent that binds to a polypeptide and alters a biological function or activity of the polypeptide. A modulator includes, without limitation, agents that reduce or eliminate a biological function or activity of a polypeptide (e.g., an “inhibitor”). For example, a modulator may inhibit a catalytic activity of a polypeptide. A modulator includes, without limitation, agents that increase or decrease binding of a polypeptide to another agent. For example, a modulator may promote binding of a polypeptide to another polypeptide. In some embodiments, a modulator of PDE3A polypeptide is DNMDP. In some other embodiments, the modulator of PDE3A polypeptide is anagrelide or zardaverine. In still other embodiments, the modulator of PDE3A polypeptide is Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, or Compound 6.

The term “prodrugs” or “prodrug” designates compounds which themselves can be biologically active or inactive, but are converted (for example metabolically or hydrolytically) into compounds according to the invention during their residence time in the body. Derivatives of the compound 6 and the salts thereof which are converted into compound 6 or a salt thereof in a biological system (bioprecursors or pro-drugs) are covered by the invention. Said biological system may be, for example, a mammalian organism, particularly a human subject. The bioprecursor is, for example, converted into the compound 6 or a salt thereof by metabolic processes.

By “reference” is meant a standard or control condition.

Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule.

By “hybridize” is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).

For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C., more preferably of at least about 37° C., and most preferably of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred: embodiment, hybridization will occur at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37° C. in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 .mu.g/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42° C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 g/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.

For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C., more preferably of at least about 42° C., and even more preferably of at least about 68° C. In a preferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

By “Solid tumors” include for example, tumors of the breast, the respiratory tract, the brain, the bones, the central and peripheral nervous system, the colon, the rectum, the anus, the reproductive organs (e.g., cervix, ovary, prostate), the gastrointestinal tract, the urogenital tract, the endocrine glands (e.g., thyroid and adrenal cortex), the thyroid gland, the parathyroid gland, the esophagus, the endometrium, the eye, the germ cells, the head and the neck, the kidney, the liver, the larynx and hypopharynx, the lung, the mesothelioma, the pancreas, the prostate, the rectum, the kidney, the small intestine, the skin, the soft tissue, the stomach, the testis, ureter, vagina and vulva and the connective tissue and metastases of these tumors. Malignant neoplasias include inherited cancers exemplified by Retinoblastoma and Wilms tumor.

-   -   “Breast tumors” that can be treated include, for example,         mammary carcinoma with positive hormone receptor status, mammary         carcinoma with negative hormone receptor status, Her-2-positive         mammary carcinoma, hormone receptor- and Her-2-negative mammary         carcinoma, BRCA-associated mammary carcinoma and inflammatory         mammary carcinoma.     -   “Tumors of the respiratory tract” that can be treated include,         for example, non-small-cell bronchial carcinoma and small-cell         bronchial carcinoma, non-small cell lung cancer, and small cell         lung cancer.     -   “Brain tumors” that can be treated include, for example, glioma,         glioblastoma, astrocytoma, meningioma and medulloblastoma.     -   “Tumors of the male reproductive organs” that can be treated         include, for example, prostate carcinoma, malignant epididymal         tumors, malignant testicular tumors and penile carcinoma.     -   “Tumors of the female reproductive organs” that can be treated         include, for example, endometrial carcinoma, cervical carcinoma,         ovarian carcinoma, vaginal carcinoma and vulvar carcinoma.     -   “Tumors of the gastrointestinal tract” that can be treated         include, for example, colorectal carcinoma, anal carcinoma,         gastric carcinoma, pancreatic carcinoma, oesophageal carcinoma,         gallbladder carcinoma, small-intestinal carcinoma, salivary         gland carcinoma, neuroendocrine tumors and gastrointestinal         stromal tumors.     -   “Tumors of the urogenital tract” that can be treated include,         for example, urinary bladder carcinoma, renal cell carcinoma,         and carcinoma of the renal pelvis and of the urinary tract.     -   “Tumors of the eye” that can be treated include, for example,         retinoblastoma and intraocular melanoma.     -   “Tumors of the liver” that can be treated include, for example,         hepatocellular carcinoma and cholangiocellular carcinoma.     -   “Tumors of the skin” that can be treated include, for example,         malignant melanoma, basalioma, spinalioma, Kaposi's sarcoma and         Merkel cell carcinoma.     -   “Tumors of the head and neck” that can be treated include, for         example, laryngeal carcinoma and carcinoma of the pharynx and of         the oral cavity.     -   “Sarcomas” that can be treated include, for example, soft tissue         sarcoma, synovial sarcoma, rhabdoid sarcoma and osteosarcoma.     -   Lymphomas that can be treated include, for example,         non-Hodgkin's lymphoma, Hodgkin's lymphoma, cutaneous lymphoma,         lymphoma of the central nervous system and AIDS-associated         lymphoma.     -   Leukaemias that can be treated include, for example, acute         myeloid leukaemia, chronic myeloid leukaemia, acute lymphatic         leukaemia, chronic lymphatic leukaemia and hair cell leukaemia.

By “substantially identical” is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.

Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e⁻³ and e⁻¹⁰⁰ indicating a closely related sequence.

By “subject” is meant a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline.

Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms “a”, “an”, and “the” are understood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

Unless specifically stated or obvious from context, as used herein, if a range is provided, the upper and lower limit are always meant to be included.

The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D show identification and characterization of 6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one (DNMDP), a potent and selective cancer cell cytotoxic agent. FIG. 1A is a scatterplot of 1924 compounds showing mean survival of TP53 mutant NCI-H1734 cells, which is a non-small cell lung cancer cell line, and TP53 wild-type A549 cells, another lung cancer cell line, after 48 hours of treatment at concentrations of 1 μM. DNMDP is indicated with a large arrowhead. Other compounds that selectively killed NCI-H1734 cells are indicated with a small arrow. Positive control staurosporine is indicated with a long arrow. FIG. 1B is a linear graph showing a panel of cell lines that was treated with the indicated concentrations of DNMDP for 48 hours. FIG. 1C is a linear graph showing the HeLa cell line that was treated with indicated concentrations of the separated enantiomers of DNMDP for 48 hours. The (R)-enantiomer had a 500-fold lower EC₅₀ compared to the (S)-enantiomer. FIG. 1D is a structure of (R)-DNMDP.

FIG. 2 shows that DNMDP selectively killed NCI-H1734 and did not affect cell viability in A549. NCI-H1734 and A549 cell lines were treated with indicated compounds and concentrations for 48 hours.

FIG. 3 shows the synthesis scheme of (R)-6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one (R)-DNMDP) and analogues. Reaction conditions are as follows: (a) Ac₂O, (91%); (b) 90% HNO₃, H₂SO₄, (19%); (c) NaOH, MeOH/H₂O, (100%), then CH₃CHO, NaBH(OAc)₃, (7%); (d) (BrCH₂CH₂)₂O, K₂CO₃, DMF, (46%); (e) CH₃CHO, NaBH₃CN, MeOH, (82%).

FIGS. 4A-4C show super-critical fluid (SCF) chromatographs of 6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one (DNMDP) (top to bottom: ES+, diode array, ES− traces). FIG. 4A are three chromatographs showing Peak 1 (CRO separation); FIG. 4B are three chromatographs showing Peak 2 (CRO separation); FIG. 4C are three chromatographs showing synthesized (R)-DNMDP (5:95 ratio peaks 1:2 by uv).

FIGS. 5A-5C show that Phosphodiesterase 3A (PDE3A) expression correlated with sensitivity to 6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one (DNMDP), but inhibition of PDE3A mediated cAMP hydrolysis did not correlate with cytotoxicity. FIG. 5A is a scatterplot showing correlation between DNMDP sensitivity and expression of 18,988 genes in 766 genomically characterized cell lines. Cell lines were treated for 72 hours with concentrations ranging from 66.4 μM-2 nM in 2-fold step dilutions. The Z-score for Pearson correlation between PDE3A expression and sensitivity to DNMDP is 8.5. FIG. 5B is a scatterplot showing results from cell lines from panel A that were treated with 480 compounds. DNMDP showed the best correlation between PDE3A expression and sensitivity. FIG. 5C is a scatterplot showing published PDE3 inhibitor IC₅₀ values and EC₅₀ values of HeLa cells treated with indicated compounds up to 10 μM for 48 hours. DNMDP IC₅₀ concentration for PDE3A inhibition was determined in FIG. 7B.

FIGS. 6A-6C show chemical structures of 6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one (DNMDP), siguazodan and levosimendan, respectively.

FIGS. 7A and 7B are graphs showing determination of Phosphodiesterase 3A (PDE3A) in vitro IC₅₀ of 6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one (DNMDP). FIG. 7A shows PDE3A in vitro inhibition with indicated concentrations of positive control trequinsin (IC₅₀ curve was performed by Caliper). FIG. 7B shows PDE3A in vitro inhibition with indicated concentrations of DNMDP (IC₅₀ curve was performed by Caliper).

FIGS. 8A and 8B are graphs showing that induction of cAMP signaling did not phenocopy cytotoxicity induced by 6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one (DNMDP). Forskolin: FSK. FIG. 8A shows cAMP concentrations that were measured 1 hour after treatment with indicated compounds and concentration in HeLa cells. FIG. 8B shows viability of HeLa cells that were treated with indicated compounds and concentrations for 48 hours.

FIGS. 9A-9C show that non-lethal Phosphodiesterase 3 (PDE3) inhibitors rescued cell death induced by 6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one (DNMDP) by competing for the binding of PDE3A. FIG. 9A is a scatterplot showing viability of HeLa cells that were treated with 1600 bioactive compounds at a concentration of 2 μM in combination with 30 nM (EC70) of DNMDP for 48 hours. The viability was calculated as a percentage of the untreated DMSO control. FIG. 9B is a linear graph showing viability of HeLa cells that were treated with DNMDP in combination with indicated concentrations of non-lethal PDE3 and pan-PDE inhibitors for 48 hours. FIG. 9C shows a SDS-PAGE gel depicting the result of affinity purification performed on 200 g of HeLa cell lysate using a DNMDP linker-analogue tethered to a solid phase with the same rescue characteristic as non-lethal PDE3 inhibitors. Indicated compounds were co-incubated with the linker-analogue. The affinity purified fraction was run on an SDS-PAGE gel and probed for PDE3A.

FIGS. 10A and 10B show the structure and rescue phenotype of linker-compound tert-butyl (R)-(2-(2-(2-(ethyl(4-(4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)phenyl)amino)ethoxy) ethoxy)ethyl)carbamate (DNMDP)-2L. FIG. 10A shows the structure of DNMDP-2L. Figure 10B is a linear graph showing the viability of HeLa cells that were treated with indicated compounds and concentrations for 48 hours.

FIGS. 11A-11C show that Phosphodiesterase 3A (PDE3A) was not essential in sensitive cell lines, but was required for relaying the cytotoxic signal. FIG. 11A is a Western blot. HeLa cells were infected with Cas9 and indicated guide RNAs (sgRNA) against PDE3A. Western blots were probed for PDE3A at indicated time points. FIG. 11B is a bar graph showing percent rescue of HeLa cells that were infected with indicated sgRNAs for two weeks and treated with 1 μM of 6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one (DNMDP) for 48 hours. Percent rescue was normalized to the Cas9-only control. FIG. 11C is a plot showing viability of cells infected with indicated sgRNAs and treated with various concentrations of 6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one (DNMDP).

FIGS. 12A and 12B are a Western blot and a graph showing that reduction of Phosphodiesterase 3A (PDE3A) protein level caused resistance to 6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one (DNMDP). In FIG. 12A HeLa cells were treated with scrambled control siRNA or a combination of four different siRNAs targeting PDE3A. Cells were lysed at indicated time-points and immunoblotted for PDE3A and Actin. FIG. 12B is a linear graph showing viability of HeLa cells that were treated with indicated concentrations of DNMDP analogue 3 for 48 hours.

FIGS. 13A-13C show that Phosphodiesterase 3A (PDE3A) immunoprecipitation in the presence of 6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one (DNMDP) revealed novel SIRT7 and SLFN12 interaction. FIG. 13A shows a schematic overview of the affinity enrichment followed by quantitative proteomics of PDE3A performed in HeLa cells. All cells were treated for four hours prior to lysis with 1 μM of indicated compounds. The presence of all compounds was maintained throughout the experiment including washing steps. FIG. 13B is a scatterplot showing log 2 ratios for proteins that were enriched in anti-PDE3A immunoprecipitates in the DMSO treated HeLa cells compared to anti-PDE3A immunoprecipitates in the presence of blocking peptide specific to the PDE3A antibody; each dot represents a protein. FIG. 13C is a scatterplot showing Log₂ ratios of changes of proteins bound to PDE3A in the presence of DNMDP versus trequinsin. Each dot represents the average of two replicates per condition for an individual protein. In all cases, the data plotted passed the Bland-Altman test with 95% confidence interval for reproducibility.

FIGS. 14A-14C show results of replicate PDE3A-protein interaction studies using PDE3A as bait under different conditions. Each scatterplot showed log₂ ratios of two replicates for proteins that were enriched by PDE3A under different conditions over enrichment by PDE3A in the presence of blocking peptide. Each dot represents the log₂ ratio for that particular protein, medium gray dots correspond to a Benjamini-Hochberg adjusted p value <0.01, light gray dots represent proteins that fall outside of the Blandt-Altman test for reproducibility within a 95% confidence interval. In FIG. 14A protein enrichment was accomplished by immunoprecipitation using anti-PDE3A. In FIG. 14B protein enrichment was accomplished by immunoprecipitation using anti-PDE3A in the presence of DNMDP. In FIG. 14C protein enrichment was accomplished by immunoprecipitation using anti-PDE3A in the presence of trequinsin.

FIGS. 15A-15E show that cell lines with dual expression of SLFN12 and PDE3A were significantly enriched for DNMDP-sensitive cell lines. FIG. 15A is a scatterplot showing mRNA robust multichip average (RMA) expression values for PDE3A and SLFN12 from the Cancer Cell Line Encyclopedia (CCLE) database (a detailed genetic characterization of a large panel of human cancer cell lines;) with sensitive cell lines indicated (Barretina et al., Nature 483, 603-607, 2012). 21 sensitive cell lines were binned in three groups of 7 based on area under the curve (AUC) rank. FIG. 15B is a bar graph showing results of a Fisher's exact test on DNMDP sensitivity of cell lines with high expression of both SLFN12 and PDE3A (RMA Log 2>5) compared to other cell lines. The top half of the bar on the right indicates melanoma cell lines. FIG. 15C is a scatterplot showing mRNA RPKM+1 expression values for PDE3A and SLFN12 from RNA sequencing data. FIG. 15D is a bar graph showing qPCR expression changes of SLFN12 in HeLa cells transduced with shSLFN12 normalized to GAPDH. FIG. 15E is a plot showing viability of HeLa cells transduced with indicated shRNA reagents and treated with indicated concentrations of DNMDP for 72 hours.

FIGS. 16A and 16B are scatter plots showing that SLFN12 expression was amongst the top genes correlating with DNMDP sensitivity. FIG. 16A shows the correlation between DNMDP sensitivity and expression of 18,988 genes in 766 genomically characterized cell lines. Cell lines were treated for 72 hours with concentrations ranging from 66.4 μM-2 nM in 2-fold step dilutions. FIG. 16B is a scatterplot showing a correlation between DNMDP sensitivity and expression of 18,988 genes in 766 genomically characterized cell lines. Expression levels were corrected for PDE3A expression as described earlier (Kim et al., Genetica 131, 151-156, 2007). Cell lines were treated for 72 hours with concentrations ranging from 66.4 μM-2 nM in 2-fold step dilutions.

FIGS. 17A-7B show that DNMDP induces apoptosis in HeLa cells. FIG. 17A is a plot showing viability of HeLa cells treated for 48 hours with indicated concentrations of DNMDP. Caspase-Glo represents Caspase 3/7 activity indicating induction of apoptosis. CellTiter-Glo reflects viability. FIG. 17B is an immunoblot. HeLa cells were treated for 36 hours with indicated compounds and concentrations. HeLa cells were harvested and immunoblotted for PARP-cleavage products, indicative of apoptosis.

FIG. 18 is a scatterplot of PDE3A mRNA expression and sensitivity to DNMDP of 766 cancer cell lines.

FIG. 19 is an immunoblot showing that DNMDP induces interaction between PDE3A and SIRT7 and SLFN12 in HeLa cells. HeLa cells were transfected with indicated plasmids and treated with indicated compounds with a final concentration of 1 μM for four hours. Endogenous PDE3A was immunoprecipitated and immunoblotted for V5 to identify novel interaction with SIRT7 and SLFN12 (upper two panels). Immunoprecipitate input was immunoblotted for PDE3A and V5 (lower two panels). V5-SLFN12 was undetectable in whole cell lysate.

FIG. 20 is an immunoblot showing confirmation of mass spectrometric results herein using affinity reagents. FIG. 20 shows that SLFN12 is required for DNMDP activity. FIG. 20 shows that DNMDP and (weakly) anagrelide, but not trequinsin, induced PDE3A and SFLN12 complex formation.

FIGS. 21A-21C show that dose-dependent PDE3A/SLFN12 complex formation correlated with cell killing potency in HeLa cells. FIG. 21A is an immunoblot showing the levels of SLFN12-V5 in cells treated with Compound 7, Compound 8 and Compound 3. FIG. 21B is a plot showing complex formation in HeLa cells, as measured by quantifying levels of SLFN12-V5 in cells treated with Compound 7, Compound 8 and Compound 3 in FIG. 21A. FIG. 21C is a plot showing cell killing in HeLa cells treated with Compound 7, Compound 8 and Compound 3. The structures of the compounds are shown below:

FIG. 22 is a set of tables showing that SLFN12 and CREB3L are lost in cells that have acquired resistance to DNMDP.

FIG. 23A is a plot showing sensitization of a DNMDP-resistant HeLa cell line by expression of SLFN12, which was competed away by the PDE3A inhibitor, trequinsin.

FIG. 23B is a plot showing sensitization of a DNMDP-resistant cell line (A549) by expression of SLFN12 or expression of SFLN12 and PDE3A.

FIG. 24 is a scatter plot showing predicted sensitivity of Leiomyosarcomas (LMS) to PDE3A modulation based on SLFN12 and PDE3A expression level.

Table 1 shows sensitivity data of 766 cancer cell lines treated with DNMDP. Cell lines were treated for 72 hours with concentrations ranging from 66.4 μM-2 nM in 2-fold step dilutions.

Table 1a shows IC₅₀ values obtained by ell proliferation results measurements for compound 6.

Table 2 shows results from panel of 19 phosphodiesterase inhibition reactions performed by Caliper. DNMDP concentration was 100 nM.

Table 3 shows RPKM values of SLFN12 and PDE3A expression in multiple healthy tissue types.

Table 4 shows that Leiomyosarcomas are predicted to be sensitive to DNMDP

Table 5 shows binding of DNMDP to PDE3A(677-1141).

Compositions and articles defined by the invention were isolated or otherwise manufactured in connection with the examples provided below. Other features and advantages of the invention will be apparent from the detailed description, and from the claims.

DETAILED DESCRIPTION

As described below, the present invention features improved methods of identifying patients having cancer (e.g the cancer types described herein) that is sensitive to treatment with a phosphodiesterase 3A (PDE3A) modulator by detecting co-expression of PDE3A and Schlafen 12 (SLFN12) polypeptides or polynucleotides in a cancer cell derived from such patients. The invention is based at least in part on the discovery that sensitivity to phosphodiesterase 3A modulators, such as 6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one, or DNMDP, in 766 cancer cell lines correlated with expression of the phosphodiesterase 3A gene, PDE3A. Like DNMDP, a subset of PDE3A inhibitors kill selected cancer cells while others do not; these cell-sparing PDE3A inhibitors instead block DNMDP induced cytotoxicity. Furthermore, PDE3A depletion leads to DNMDP resistance. DNMDP binding to PDE3A promotes an interaction between PDE3A and Sirtuin 7 (SIRT7) and Schlafen 12 (SLFN12), suggesting a neomorphic activity, and SLFN12 and PDE3A co-expression correlated with DNMDP sensitivity. These results indicate that PDE3A modulators are promising cancer therapeutic agents and demonstrate the power of predictive chemogenomics in small-molecule discovery and target-identification.

Accordingly, the invention provides methods of selecting a subject as having a cancer that responds to a PDE3A modulator, where the selection method involves detecting co-expression of PDE3A and Schlafen 12 (SLFN12) polypeptides or polynucleotides, in a cancer cell derived from such subjects.

In one particular embodiment, expression of CREB3L1 or SLFN12 polynucleotide or polypeptide is reduced or is undetectable in a cancer cell that has acquired resistance to a PDE3A modulator.

PDE3A Modulator

The identification of PDE3A modulators was made in connection with a phenotypic screen designed to identify cytotoxic small molecules in a mutant tp53 background. A predictive chemogenomics approach complements target-driven drug development programs, which consists of extensive in vitro and in vivo target validation, and can also be referred to as reverse chemogenomics (Zheng et al., Curr Issues Mol Biol 4, 33-43, 2002). Many U.S. Food and Drug Administration (FDA)-approved targeted therapies have been developed this way, among them small-molecule kinase inhibitors that target oncogenic somatic driver mutations (Moffat et al., Nat Rev Drug Discov 13, 588-602, 2014). However, the discovery and development of targeted therapies is often hampered by limitations in knowledge of the biological function of the target, its mechanism of action, and the available chemical matter to selectively inhibit the target.

Phenotypic screening can discover novel targets for cancer therapy whose specific molecular mechanism is often elucidated by future studies (Swinney et al., Nat Rev Drug Discov 10, 507-519, 2011). In recent years, two classes of anti-cancer drugs found by unbiased phenotypic screening efforts have been approved by the FDA. Lenalidomide and pomalidomide were found to be modulators of an E3-ligase that alter the affinity of its target, leading to degradation of lineage specific transcription factors (Krönke et al., Science 343, 301-305, 2014; Lu et al., Science 343, 305-309, 2014), whereas romidepsin and vorinostat were later identified as histone deacetylase (HDAC) inhibitors (Moffat et al., Nat Rev Drug Discov 13, 588-602, 2014; Nakajima et al., Exp. Cell Res. 241, 126-133, 1998, Marks et al., Nat Biotechnol 25, 84-90, 2007).

Tumor suppressor alterations are suitable targets for phenotypic screening as they are not directly targetable with small molecules, although synthetic lethal approaches such as olaparib treatment of BRCA1/BRCA2 mutant cancers have proven to be effective. According to current knowledge, the tp53 tumor suppressor gene is the most frequently mutated across human cancer, with somatic mutations detected in 36% of 4742 cancers subjected to whole exome sequencing. Despite many attempts, no compounds that selectively kill tp53 mutant cells have been identified.

A phenotypic screen developed to identify small molecules causing synthetic lethality in tp3 mutant cancer cells enabled the serendipitous discovery of a class of cancer-selective cytotoxic agents which act as modulators of phosphodiesterase 3A (PDE3A), as described herein below. Cyclic nucleotide phosphodiesterases catalyze the hydrolysis of second messenger molecules cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), and are important in many physiological processes. Several phosphodiesterase inhibitors have been approved for clinical treatment, including PDE3 inhibitors milrinone, cilostazol, and levosimendan for cardiovascular indications and inhibition of platelet coagulation, as well as the PDE3 inhibitor anagrelide for thrombocythemia. Further PDE3A inhibitors are known from WO 2014/164704. PDE5 inhibitors, e.g. vardenafil, are used for smooth muscle disorders including erectile dysfunction and pulmonary arterial hypertension, and the PDE4 inhibitor roflumilast reduces exacerbations from chronic obstructive pulmonary disease (COPD).

Phosphodiesterase inhibitors act by direct inhibition of their targets or by allosteric modulation; for example, structural analysis of PDE4 has led to the design of PDE4D and PDE4B allosteric modulators (Burgin et al., Nat Biotechnol 28, 63-70, 2010; Gurney et al., Neurotherapeutics 12, 49-56, 2015). The data provided herein below indicates that the cancer cytotoxic phosphodiesterase modulator DNMDP likely acts through a similar allosteric mechanism.

Accordingly, the invention provides methods for identifying subjects that have a malignancy that is likely to respond to PDE3A modulator treatment based on the level of PDE3A and SLFN12 expression in a subject biological sample comprising a cancer cell. In some embodiments, the PDE3A modulator is DNMDP. In some other embodiments, the PDE3A modulator is anagrelide or zardaverine. In still other embodiments, the PDE3A modulator is Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, or Compound 6.

In particular embodiments, the invention provides methods for identifying subjects that have a malignancy that is resistant to PDE3A modulator treatment based on a loss or reduction in the level of CREB3L1 or SLFN12 expression relative to a reference.

Compound Forms and Salts

The compounds of the present invention include the compounds themselves, as well as their salts and their prodrugs, if applicable. A salt, for example, can be formed between an anion and a positively charged substituent (e.g., amino) on a compound described herein. Suitable anions include chloride, bromide, iodide, sulfate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, and acetate. Likewise, a salt can also be formed between a cation and a negatively charged substituent (e.g., carboxylate) on a compound described herein. Suitable cations include sodium ion, potassium ion, magnesium ion, calcium ion, and an ammonium cation such as tetramethylammonium ion. Examples of prodrugs include C₁₋₆ alkyl esters of carboxylic acid groups, which, upon administration to a subject, are capable of providing active compounds.

Pharmaceutically acceptable salts of the compounds of the present disclosure include those derived from pharmaceutically acceptable inorganic and organic acids and bases. As used herein, the term “pharmaceutically acceptable salt” refers to a salt formed by the addition of a pharmaceutically acceptable acid or base to a compound disclosed herein. As used herein, the phrase “pharmaceutically acceptable” refers to a substance that is acceptable for use in pharmaceutical applications from a toxicological perspective and does not adversely interact with the active ingredient.

A suitable pharmaceutically acceptable salt of the compounds of the present invention may be, for example, an acid-addition salt of a compound of the present invention bearing a nitrogen atom, in a chain or in a ring, for example, which is sufficiently basic, such as an acid-addition salt with an inorganic acid, or “mineral acid”, such as hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfamic, bisulfuric, phosphoric, or nitric acid, for example, or with an organic acid, such as formic, acetic, acetoacetic, pyruvic, trifluoroacetic, propionic, butyric, hexanoic, heptanoic, undecanoic, lauric, benzoic, salicylic, 2-(4-hydroxybenzoyl)-benzoic, camphoric, cinnamic, cyclopentanepropionic, digluconic, 3-hydroxy-2-naphthoic, nicotinic, pamoic, pectinic, 3-phenylpropionic, pivalic, 2-hydroxyethanesulfonic, itaconic, trifluoromethanesulfonic, dodecylsulfuric, ethanesulfonic, benzenesulfonic, para-toluenesulfonic, methanesulfonic, 2-naphthalenesulfonic, naphthalinedisulfonic, camphorsulfonic acid, citric, tartaric, stearic, lactic, oxalic, malonic, succinic, malic, adipic, alginic, maleic, fumaric, D-gluconic, mandelic, ascorbic, glucoheptanoic, glycerophosphoric, aspartic, sulfosalicylic, or thiocyanic acid, for example.

Further examples of suitable acid salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, thiocyanate, tosylate and undecanoate. Other acids, such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds of the present invention and their pharmaceutically acceptable acid addition salts.

Further, another suitably pharmaceutically acceptable salt of a compound 1-6, especially of compound 6, which is sufficiently acidic, is an alkali metal salt, for example a sodium or potassium salt, an alkaline earth metal salt, for example a calcium, magnesium or strontium salt, or an aluminium or a zinc salt, or an ammonium salt derived from ammonia or from an organic primary, secondary or tertiary amine having 1 to 20 carbon atoms, such as ethylamine, diethylamine, triethylamine, ethyldiisopropylamine, monoethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, dimethylaminoethanol, diethylaminoethanol, tris(hydroxymethyl)aminomethane, procaine, dibenzylamine, N-methylmorpholine, arginine, lysine, 1,2-ethylenediamine, N-methylpiperidine, N-methyl-glucamine, N,N-dimethyl-glucamine, N-ethyl-glucamine, 1,6-hexanediamine, glucosamine, sarcosine, serinol, 2-amino-1,3-propanediol, 3-amino-1,2-propanediol, 4-amino-1,2,3-butanetriol, or a salt with a quarternary ammonium ion having 1 to 20 carbon atoms, such as tetramethylammonium, tetraethylammonium, tetra(n-propyl)ammonium, tetra(n-butyl)ammonium, N-benzyl-N,N,N-trimethylammonium, choline or benzalkonium.

In certain embodiments salts are derived from appropriate bases include alkali metal (e.g., sodium), alkaline earth metal (e.g., magnesium), ammonium and N-(alkyl)₄ salts. The present invention also envisions the quaternization of any basic nitrogen-containing groups of the compounds disclosed herein. Water or oil-soluble or dispersible products may be obtained by such quaternization. Salt forms of the compounds of any of the formulae herein can be amino acid salts of carboxyl groups (e.g., L-arginine, -lysine, -histidine salts).

Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418; Journal of Pharmaceutical Science, 66, 2 (1977); and “Pharmaceutical Salts: Properties, Selection, and Use A Handbook; Wermuth, C. G. and Stahl, P. H. (eds.) Verlag Helvetica Chimica Acta, Zurich, 2002 [ISBN 3-906390-26-8] each of which is incorporated herein by reference in their entireties. Those skilled in the art will further recognise that it is possible for acid addition salts of the claimed compounds to be prepared by reaction of the compounds with the appropriate inorganic or organic acid via any of a number of known methods. Alternatively, alkali and alkaline earth metal salts of acidic compounds of the present invention are prepared by reacting the compounds of the present invention with the appropriate base via a variety of known methods.

The present invention includes all possible salts of the compounds of the present invention as single salts, or as any mixture of said salts, in any ratio.

The neutral forms of the compounds may be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for the purposes of the present invention.

In addition to salt forms, the present invention provides compounds which are in a prodrug form. Prodrugs of the compounds described herein are those compounds that undergo chemical changes under physiological conditions to provide the compounds of the present invention. Additionally, prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds of the present invention when placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent. Prodrugs are often useful because, in some situations, they may be easier to administer than the parent drug. They may, for instance, be more bioavailable by oral administration than the parent drug. The prodrug may also have improved solubility in pharmacological compositions over the parent drug. A wide variety of prodrug derivatives are known in the art, such as those that rely on hydrolytic cleavage or oxidative activation of the prodrug. An example, without limitation, of a prodrug would be a compound of the present invention which is administered as an ester (the “prodrug”), but then is metabolically hydrolyzed to the carboxylic acid, the active entity. Additional examples include peptidyl derivatives of a compound of the present invention.

The present invention also includes various hydrate and solvate forms of the compounds.

The compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example tritium (3H), iodine-125 (¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention. particularly deuterium-containing compounds.

The term “Isotopic variant” of a compound or a reagent is defined as a compound exhibiting an unnatural proportion of one or more of the isotopes that constitute such a compound.

The expression “unnatural proportion” means a proportion of such isotope which is higher than its natural abundance. The natural abundances of isotopes to be applied in this context are described in “Isotopic Compositions of the Elements 1997”, Pure Appl. Chem., 70(1), 217-235, 1998.

Examples of such isotopes include stable and radioactive isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, bromine and iodine, such as ²H (deuterium), ³H (tritium), ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁷O, ¹⁸O, ³²P, ³³P, ³³S, ³⁴S, ³⁵S, ³⁶S, ¹⁸F, ³⁶Cl, ⁸²Br, ¹²³I, ¹²⁴I, ¹²⁵I, ¹²⁹I and ¹³¹I, respectively.

With respect to the treatment and/or prophylaxis of the disorders specified herein the isotopic variant(s) of the compounds 1-6, especially of compound 6, preferably contain deuterium (“deuterium-containing”). Isotopic variants of the compounds 1-6, especially of compound 6, in which one or more radioactive isotopes, such as ³H or ¹⁴C, are incorporated are useful e.g. in drug and/or substrate tissue distribution studies. These isotopes are particularly preferred for the ease of their incorporation and detectability. Positron emitting isotopes such as ¹⁸F or ¹¹C may be incorporated into a compound 1-6, especially in compound 6. These isotopic variants of the compounds 1-6 are useful for in vivo imaging applications. Deuterium-containing and ¹³C-containing compounds 1-6 can be used in mass spectrometry analyses in the context of preclinical or clinical studies.

Isotopic variants of the compounds 1-6 can generally be prepared by methods known to a person skilled in the art, such as those described in the schemes and/or examples herein, by substituting a reagent for an isotopic variant of said reagent, preferably for a deuterium-containing reagent. Depending on the desired sites of deuteration, in some cases deuterium from D₂O can be incorporated either directly into the compounds or into reagents that are useful for synthesizing such compounds. Deuterium gas is also a useful reagent for incorporating deuterium into molecules. Catalytic deuteration of olefinic bonds and acetylenic bonds is a rapid route for incorporation of deuterium. Metal catalysts (i.e. Pd, Pt, and Rh) in the presence of deuterium gas can be used to directly exchange deuterium for hydrogen in functional groups containing hydrocarbons. A variety of deuterated reagents and synthetic building blocks are commercially available from companies such as for example C/D/N Isotopes, Quebec, Canada; Cambridge Isotope Laboratories Inc., Andover, Mass., USA; and CombiPhos Catalysts, Inc., Princeton, N.J., USA.

The term “deuterium-containing compounds 1-6” is defined as a compound, in which one or more hydrogen atom(s) is/are replaced by one or more deuterium atom(s) and in which the abundance of deuterium at each deuterated position of anyone of the compound 1-6 is higher than the natural abundance of deuterium, which is about 0.015%. Particularly, in anyone of a deuterium-containing compound 1-6 the abundance of deuterium at each deuterated position of the compound is higher than 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80%, preferably higher than 90%, 95%, 96% or 97%, even more preferably higher than 98% or 99% at said position(s). It is understood that the abundance of deuterium at each deuterated position is independent of the abundance of deuterium at other deuterated position(s).

The selective incorporation of one or more deuterium atom(s) into anyone of a compound 1-6 may alter the physicochemical properties (such as for example acidity [C. L. Perrin, et al., J. Am. Chem. Soc., 2007, 129, 4490], basicity [C. L. Perrin et al., J. Am. Chem. Soc., 2005, 127, 9641], lipophilicity [B. Testa et al., Int. J. Pharm., 1984, 19(3), 271]) and/or the metabolic profile of the molecule and may result in changes in the ratio of parent compound to metabolites or in the amounts of metabolites formed. Such changes may result in certain therapeutic advantages and hence may be preferred in some circumstances. Reduced rates of metabolism and metabolic switching, where the ratio of metabolites is changed, have been reported (A. E. Mutlib et al., Toxicol. Appl. Pharmacol., 2000, 169, 102). These changes in the exposure to parent drug and metabolites can have important consequences with respect to the pharmacodynamics, tolerability and efficacy of a deuterium-containing compound of general formula (I). In some cases deuterium substitution reduces or eliminates the formation of an undesired or toxic metabolite and enhances the formation of a desired metabolite (e.g. Nevirapine: A. M. Sharma et al., Chem. Res. Toxicol., 2013, 26, 410; Efavirenz: A. E. Mutlib et al., Toxicol. Appl. Pharmacol., 2000, 169, 102). In other cases the major effect of deuteration is to reduce the rate of systemic clearance. As a result, the biological half-life of the compound is increased. The potential clinical benefits would include the ability to maintain similar systemic exposure with decreased peak levels and increased trough levels. This could result in lower side effects and enhanced efficacy, depending on the particular compound's pharmacokinetic/pharmacodynamic relationship. ML-337 (C. J. Wenthur et al., J. Med. Chem., 2013, 56, 5208) and Odanacatib (K. Kassahun et al., WO2012/112363) are examples for this deuterium effect. Still other cases have been reported in which reduced rates of metabolism result in an increase in exposure of the drug without changing the rate of systemic clearance (e.g. Rofecoxib: F. Schneider et al., Arzneim. Forsch./Drug. Res., 2006, 56, 295; Telaprevir: F. Maltais et al., J. Med. Chem., 2009, 52, 7993).

Deuterated drugs showing this effect may have reduced dosing requirements (e.g. lower number of doses or lower dosage to achieve the desired effect) and/or may produce lower metabolite loads.

The compounds 1-6 may have multiple potential sites of attack for metabolism. To optimize the above-described effects on physicochemical properties and metabolic profile, deuterium-containing compounds 1-6 having a certain pattern of one or more deuterium-hydrogen exchange(s) can be selected. Particularly, the deuterium atom(s) of deuterium-containing compound(s) 1-6 is/are attached to a carbon atom and/or is/are located at those positions of the compound 1-6, which are sites of attack for metabolizing enzymes such as e.g. cytochrome P450.

Pharmaceutical Composition

It is possible for the compounds 1-6, especially for compound 6, to have systemic and/or local activity. For this purpose, they can be administered in a suitable manner, such as, for example, via the oral, parenteral, pulmonary, nasal, sublingual, lingual, buccal, rectal, vaginal, dermal, transdermal, conjunctival, otic route or as an implant or stent.

For these administration routes, it is possible for the compounds 1-6 to be administered in suitable administration forms.

For oral administration, it is possible to formulate the compounds 1-6 to dosage forms known in the art that deliver the compounds of the invention rapidly and/or in a modified manner, such as, for example, tablets (uncoated or coated tablets, for example with enteric or controlled release coatings that dissolve with a delay or are insoluble), orally-disintegrating tablets, films/wafers, films/lyophylisates, capsules (for example hard or soft gelatine capsules), sugar-coated tablets, granules, pellets, powders, emulsions, suspensions, aerosols or solutions. It is possible to incorporate the compounds 1-6 in crystalline and/or amorphised and/or dissolved form into said dosage forms.

Parenteral administration can be effected with avoidance of an absorption step (for example intravenous, intraarterial, intracardial, intraspinal or intralumbal) or with inclusion of absorption (for example intramuscular, subcutaneous, intracutaneous, percutaneous or intraperitoneal). Administration forms which are suitable for parenteral administration are, inter alia, preparations for injection and infusion in the form of solutions, suspensions, emulsions, lyophylisates or sterile powders.

Examples which are suitable for other administration routes are pharmaceutical forms for inhalation [inter alia powder inhalers, nebulizers], nasal drops, nasal solutions, nasal sprays; tablets/films/wafers/capsules for lingual, sublingual or buccal administration; suppositories; eye drops, eye ointments, eye baths, ocular inserts, ear drops, ear sprays, ear powders, ear-rinses, ear tampons; vaginal capsules, aqueous suspensions (lotions, mixturae agitandae), lipophilic suspensions, emulsions, ointments, creams, transdermal therapeutic systems (such as, for example, patches), milk, pastes, foams, dusting powders, implants or stents.

The compounds according to the invention can be incorporated into the stated administration forms. This can be effected in a manner known per se by mixing with pharmaceutically suitable excipients. Pharmaceutically suitable excipients include, inter alia,

-   -   fillers and carriers (for example cellulose, microcrystalline         cellulose (such as, for example, Avicel®), lactose, mannitol,         starch, calcium phosphate (such as, for example, Di-Cafos®)),     -   ointment bases (for example petroleum jelly, paraffins,         triglycerides, waxes, wool wax, wool wax alcohols, lanolin,         hydrophilic ointment, polyethylene glycols),     -   bases for suppositories (for example polyethylene glycols, cacao         butter, hard fat),     -   solvents (for example water, ethanol, isopropanol, glycerol,         propylene glycol, medium chain-length triglycerides fatty oils,         liquid polyethylene glycols, paraffins),     -   surfactants, emulsifiers, dispersants or wetters (for example         sodium dodecyl sulfate), lecithin, phospholipids, fatty alcohols         (such as, for example, Lanette®), sorbitan fatty acid esters         (such as, for example, Span®), polyoxyethylene sorbitan fatty         acid esters (such as, for example, Tween®), polyoxyethylene         fatty acid glycerides (such as, for example, Cremophor®),         polyoxethylene fatty acid esters, polyoxyethylene fatty alcohol         ethers, glycerol fatty acid esters, poloxamers (such as, for         example, Pluronic®),     -   buffers, acids and bases (for example phosphates, carbonates,         citric acid, acetic acid, hydrochloric acid, sodium hydroxide         solution, ammonium carbonate, trometamol, triethanolamine),     -   isotonicity agents (for example glucose, sodium chloride),     -   adsorbents (for example highly-disperse silicas),     -   viscosity-increasing agents, gel formers, thickeners and/or         binders (for example polyvinylpyrrolidone, methylcellulose,         hydroxypropylmethylcellulose, hydroxypropyl-cellulose,         carboxymethylcellulose-sodium, starch, carbomers, polyacrylic         acids (such as, for example, Carbopol®); alginates, gelatine),     -   disintegrants (for example modified starch,         carboxymethylcellulose-sodium, sodium starch glycolate (such as,         for example, Explotab®), cross-linked polyvinylpyrrolidone,         croscarmellose-sodium (such as, for example, AcDiSol®)),     -   flow regulators, lubricants, glidants and mould release agents         (for example magnesium stearate, stearic acid, talc,         highly-disperse silicas (such as, for example, Aerosil)),     -   coating materials (for example sugar, shellac) and film formers         for films or diffusion membranes which dissolve rapidly or in a         modified manner (for example polyvinylpyrrolidones (such as, for         example, Kollidon®), polyvinyl alcohol,         hydroxypropylmethylcellulose, hydroxypropylcellulose,         ethylcellulose, hydroxypropyl-methylcellulose phthalate,         cellulose acetate, cellulose acetate phthalate, polyacrylates,         polymethacrylates such as, for example, Eudragit®)),     -   capsule materials (for example gelatine,         hydroxypropylmethylcellulose),     -   synthetic polymers (for example polylactides, polyglycolides,         polyacrylates, polymethacrylates (such as, for example,         Eudragit®), polyvinylpyrrolidones (such as, for example,         Kollidon®), polyvinyl alcohols, polyvinyl acetates, polyethylene         oxides, polyethylene glycols and their copolymers and         blockcopolymers),     -   plasticizers (for example polyethylene glycols, propylene         glycol, glycerol, triacetine, triacetyl citrate, dibutyl         phthalate),     -   penetration enhancers,     -   stabilisers (for example antioxidants such as, for example,         ascorbic acid, ascorbyl palmitate, sodium ascorbate,         butylhydroxyanisole, butylhydroxytoluene, propyl gallate),     -   preservatives (for example parabens, sorbic acid, thiomersal,         benzalkonium chloride, chlorhexidine acetate, sodium benzoate),     -   colourants (for example inorganic pigments such as, for example,         iron oxides, titanium dioxide),     -   flavourings, sweeteners, flavour- and/or odour-masking agents.

The present invention furthermore relates to a pharmaceutical composition which comprise at least one compound 1-6, especially compound 6, conventionally together with one or more pharmaceutically suitable excipient(s), and to their use according to the present invention.

Combinations

In accordance with another aspect, the present invention covers pharmaceutical combinations, in particular medicaments, comprising at least one of the compounds 1-6, especially compound 6 and at least one or more further active ingredients, in particular for the treatment and/or prophylaxis of a hyperproliferative disease, especially cancer.

Particularly, the present invention covers a pharmaceutical combination, which comprises:

-   -   one or more first active ingredients, in particular one of the         compounds 1-6, especially compound 6, as defined supra, and     -   one or more further active ingredients, in particular a         hyperproliferative disease, especially cancer

The term “combination” in the present invention is used as known to persons skilled in the art, it being possible for said combination to be a fixed combination, a non-fixed combination or a kit-of-parts.

A “fixed combination” in the present invention is used as known to persons skilled in the art and is defined as a combination wherein, for example, a first active ingredient, such as one or more of compounds 1-6, and a further active ingredient are present together in one unit dosage or in one single entity. One example of a “fixed combination” is a pharmaceutical composition wherein a first active ingredient and a further active ingredient are present in admixture for simultaneous administration, such as in a formulation. Another example of a “fixed combination” is a pharmaceutical combination wherein a first active ingredient and a further active ingredient are present in one unit without being in admixture.

A non-fixed combination or “kit-of-parts” in the present invention is used as known to persons skilled in the art and is defined as a combination wherein a first active ingredient and a further active ingredient are present in more than one unit. One example of a non-fixed combination or kit-of-parts is a combination wherein the first active ingredient and the further active ingredient are present separately. It is possible for the components of the non-fixed combination or kit-of-parts to be administered separately, sequentially, simultaneously, concurrently or chronologically staggered.

The compounds of the present invention can be administered as the sole pharmaceutical agent or in combination with one or more other pharmaceutically active ingredients where the combination causes no unacceptable adverse effects. The present invention also covers such pharmaceutical combinations. For example, the compounds of the present invention can be combined with known anticancer agents and agents ameliorating potential side effects these anticancer agents may have. Examples of these agents include:

131I-chTNT, abarelix, abiraterone, aclarubicin, adalimumab, ado-trastuzumab emtansine, afatinib, aflibercept, aldesleukin, alectinib, alemtuzumab, alendronic acid, alitretinoin, altretamine, amifostine, aminoglutethimide, hexyl aminolevulinate, amrubicin, amsacrine, anastrozole, ancestim, anethole dithiolethione, anetumab ravtansine, angiotensin II, antithrombin III, aprepitant, arcitumomab, arglabin, arsenic trioxide, asparaginase, atezolizumab, axitinib, azacitidine, basiliximab, belotecan, bendamustine, besilesomab, belinostat, bevacizumab, bexarotene, bicalutamide, bisantrene, bleomycin, blinatumomab, bortezomib, buserelin, bosutinib, brentuximab vedotin, busulfan, cabazitaxel, cabozantinib, calcitonine, calcium folinate, calcium levofolinate, capecitabine, capromab, carbamazepine carboplatin, carboquone, carfilzomib, carmofur, carmustine, catumaxomab, celecoxib, celmoleukin, ceritinib, cetuximab, chlorambucil, chlormadinone, chlormethine, cidofovir, cinacalcet, cisplatin, cladribine, clodronic acid, clofarabine, cobimetinib, copanlisib, crisantaspase, crizotinib, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daratumumab, darbepoetin alfa, dabrafenib, dasatinib, daunorubicin, decitabine, degarelix, denileukin diftitox, denosumab, depreotide, deslorelin, dianhydrogalactitol, dexrazoxane, dibrospidium chloride, dianhydrogalactitol, diclofenac, dinutuximab, docetaxel, dolasetron, doxifluridine, doxorubicin, doxorubicin+estrone, dronabinol, eculizumab, edrecolomab, elliptinium acetate, elotuzumab, eltrombopag, endostatin, enocitabine, enzalutamide, epirubicin, epitiostanol, epoetin alfa, epoetin beta, epoetin zeta, eptaplatin, eribulin, erlotinib, esomeprazole, estradiol, estramustine, ethinylestradiol, etoposide, everolimus, exemestane, fadrozole, fentanyl, filgrastim, fluoxymesterone, floxuridine, fludarabine, fluorouracil, flutamide, folinic acid, formestane, fosaprepitant, fotemustine, fulvestrant, gadobutrol, gadoteridol, gadoteric acid meglumine, gadoversetamide, gadoxetic acid, gallium nitrate, ganirelix, gefitinib, gemcitabine, gemtuzumab, Glucarpidase, glutoxim, GM-CSF, goserelin, granisetron, granulocyte colony stimulating factor, histamine dihydrochloride, histrelin, hydroxycarbamide, I-125 seeds, lansoprazole, ibandronic acid, ibritumomab tiuxetan, ibrutinib, idarubicin, ifosfamide, imatinib, imiquimod, improsulfan, indisetron, incadronic acid, ingenol mebutate, interferon alfa, interferon beta, interferon gamma, iobitridol, iobenguane (123I), iomeprol, ipilimumab, irinotecan, Itraconazole, ixabepilone, ixazomib, lanreotide, lansoprazole, lapatinib, Iasocholine, lenalidomide, lenvatinib, lenograstim, lentinan, letrozole, leuprorelin, levamisole, levonorgestrel, levothyroxine sodium, lisuride, lobaplatin, lomustine, lonidamine, masoprocol, medroxyprogesterone, megestrol, melarsoprol, melphalan, mepitiostane, mercaptopurine, mesna, methadone, methotrexate, methoxsalen, methylaminolevulinate, methylprednisolone, methyltestosterone, metirosine, mifamurtide, miltefosine, miriplatin, mitobronitol, mitoguazone, mitolactol, mitomycin, mitotane, mitoxantrone, mogamulizumab, molgramostim, mopidamol, morphine hydrochloride, morphine sulfate, nabilone, nabiximols, nafarelin, naloxone+pentazocine, naltrexone, nartograstim, necitumumab, nedaplatin, nelarabine, neridronic acid, netupitant/palonosetron, nivolumab, pentetreotide, nilotinib, nilutamide, nimorazole, nimotuzumab, nimustine, nintedanib, nitracrine, nivolumab, obinutuzumab, octreotide, ofatumumab, olaparib, olaratumab, omacetaxine mepesuccinate, omeprazole, ondansetron, oprelvekin, orgotein, orilotimod, osimertinib, oxaliplatin, oxycodone, oxymetholone, ozogamicine, p53 gene therapy, paclitaxel, palbociclib, palifermin, palladium-103 seed, palonosetron, pamidronic acid, panitumumab, panobinostat, pantoprazole, pazopanib, pegaspargase, PEG-epoetin beta (methoxy PEG-epoetin beta), pembrolizumab, pegfilgrastim, peginterferon alfa-2b, pembrolizumab, pemetrexed, pentazocine, pentostatin, peplomycin, Perflubutane, perfosfamide, Pertuzumab, picibanil, pilocarpine, pirarubicin, pixantrone, plerixafor, plicamycin, poliglusam, polyestradiol phosphate, polyvinylpyrrolidone+sodium hyaluronate, polysaccharide-K, pomalidomide, ponatinib, porfimer sodium, pralatrexate, prednimustine, prednisone, procarbazine, procodazole, propranolol, quinagolide, rabeprazole, racotumomab, radium-223 chloride, radotinib, raloxifene, raltitrexed, ramosetron, ramucirumab, ranimustine, rasburicase, razoxane, refametinib, regorafenib, risedronic acid, rhenium-186 etidronate, rituximab, rolapitant, romidepsin, romiplostim, romurtide, roniciclib, samarium (153Sm) lexidronam, sargramostim, satumomab, secretin, siltuximab, sipuleucel-T, sizofiran, sobuzoxane, sodium glycididazole, sonidegib, sorafenib, stanozolol, streptozocin, sunitinib, talaporfin, talimogene laherparepvec, tamibarotene, tamoxifen, tapentadol, tasonermin, teceleukin, technetium (99mTc) nofetumomab merpentan, 99mTc-HYNIC-[Tyr3]-octreotide, tegafur, tegafur+gimeracil+oteracil, temoporfin, temozolomide, temsirolimus, teniposide, testosterone, tetrofosmin, thalidomide, thiotepa, thymalfasin, thyrotropin alfa, tioguanine, tocilizumab, topotecan, toremifene, tositumomab, trabectedin, trametinib, tramadol, trastuzumab, trastuzumab emtansine, treosulfan, tretinoin, trifluridine+tipiracil, trilostane, triptorelin, trametinib, trofosfamide, thrombopoietin, tryptophan, ubenimex, valatinib, valrubicin, vandetanib, vapreotide, vemurafenib, vinblastine, vincristine, vindesine, vinflunine, vinorelbine, vismodegib, vorinostat, vorozole, yttrium-90 glass microspheres, zinostatin, zinostatin stimalamer, zoledronic acid, zorubicin.

Utility

Compound 6 is a PDE3A inhibitor and thus according to fact that targeting cancer with phosphodiesterase inhibitors might be a promising approach compound 6 is useful for the treatment of cancer.

A further aspect of the invention is compound 6 for use in the treatment of hyperproliferative diseases.

A further aspect of the invention is the compound 6 for use in the treatment of hyperproliferative diseases are hematopoietic hyperproliferative diseases including polycythemia vera, essential thrombocytosis, primary myelofibrosis, and others.

A further aspect is the method of prophylaxis and/or treatment of hyperproliferative diseaes comprising administering an effective amount of one or more compound(s) of compound 6, especially a method of treatment of a hyperproliferative disease.

The compounds 6 are also suitable for prophylaxis and/or treatment of benign hyperproliferative diseases, for example endometriosis, leiomyoma and benign prostate hyperplasia.

Thus a further aspect is that the hyperproliferative disease is a benign hyperproliferative disease.

Another aspect of the present invention is a compound 6 for use in the treatment of cancer. They are particular useful in treating metastatic or malignant tumors.

Thus another aspect of the invention is a method of treatment of cancer comprising administering an effective amount of at least one compound 6.

A further aspect of the invention is a method of treatment of metastatic or malignant tumors comprising administering an effective amount of compound 6.

Another aspect of the invention is the use of compound 6 for the treatment of solid tumors.

A further aspect of the invention is the compound 6 for use in the treatment of solid tumors.

A further aspect of the invention is a method of treatment of solid tumors comprising administering an effective amount of compound 6.

A further aspect of the invention is the use of compound 6 for the treatment of solid tumors that can be treated as tumors of the breast, the respiratory tract, the brain, the bones, the central and peripheral nervous system, the colon, the rectum, the anus, the reproductive organs (e.g., cervix, ovary, prostate), the gastrointestinal tract (including gastrointestinal stromal tumors), the urogenital tract, the endocrine glands (e.g., thyroid and adrenal cortex), the thyroid gland, the parathyroid gland, the esophagus, the endometrium, the eye, the germ cells, the head and the neck, the kidney, the liver, the larynx and hypopharynx, the lung, the mesothelioma, the pancreas, the prostate, the rectum, the kidney, the small intestine, the skin, the soft tissue, the stomach, the testis, ureter, vagina and vulva and the connective tissue and metastases of these tumors. Malignant neoplasias include inherited cancers exemplified by Retinoblastoma and Wilms tumor.

Still another aspect of the invention is a method of treatment of the tumors mentioned above comprising administering an effective amount of compound 6.

Another aspect of the invention is the use of compound 6 for the treatment of hematological tumors.

A further aspect of the invention is the compound 6 for use in the treatment of hematological tumors.

A further aspect of the invention is a method of treatment of hematological tumors comprising administering an effective amount of compound 6.

Another aspect of the invention is the use of compound 6 for the treatment of cancer whereby the cancer type is a bone, breast, cervical, colon, endometrium, gastrointestinal stromal tumor (GIST), head and neck (especially head, more specifically glioma, glioblastoma), hematopoetic, kidney, leiomyosarcoma, liver, lung, lymphoid, melanoma ovarian, pancreas, prostate, soft-tissue sarcoma, thyroid cancer, urinary tract cancer.

Still another aspect of the invention is the use of compound 6 for the treatment of melanoma, adenocarcinoma, breast, cervical, endometrium, glioblastoma, hematopoetic/lymphoid, kidney, leiomyosarcoma, liver, lung, ovarian, pancreas, soft-tissue sarcoma, thyroid, or urinary tract cancer.

Another aspect of the invention is the use of compound 6 for the treatment of cancer whereby the cancer type is a melanoma, endometrium, lung, hematopoetic, lymphoid, ovarian, cervical, soft-tissue sarcoma, leiomyosarcoma, urinary tract, pancreas, thyroid cancer.

Yet another aspect of the invention is the use of compound 6 for the treatment of skin cancer (especially melanoma), lung cancer (especially lung adenocarcinoma) and cervical cancer.

A further aspect of the invention is the use of compound 6 for the treatment of cancer of bone, central nervous system (especially glioblastoma multiforme and glioma), colon, hematopoietic and lymphoid tissue (especially erythroleucemia and T-cell lymphoma), liver, lung (especially lung adenocarcinoma and small cell lung cancer (SCLC)), ovary, skin (especially melanoma).

Diagnostics

The present invention features diagnostic assays for the characterization of cancer. In one embodiment, levels of PDE3A, Schlafen 12 (SLFN12), or CREB3L1 polynucleotides or polypeptides are measured in a subject sample and used as an indicator of cancer that is responsive to treatment with a PDE3A modulator. In another embodiment, the level of a CREB3L1 polynucleotide or polypeptide is measured in a biological sample of the subject. A loss of or reduction in the level of CREB3L1 or SLFN12 polynucleotide or polypeptide expression in a biological sample of the subject (e.g., a biological sample comprising a cancer cell) relative to a reference indicates that the cancer is resistant to treatment with a PDE3A modulator. Levels of PDE3A, Schlafen 12 and/or CREB3L1 polynucleotides may be measured by standard methods, such as quantitative PCR, RNA sequencing, Northern Blot, microarray, mass spectrometry, and in situ hybridization. Standard methods may be used to measure levels of PDE3A, Schlafen 12, and/or CREB3L1 polypeptides in a biological sample derived from a tumor. Such methods include immunoassay, ELISA, western blotting using an antibody that binds PDE3A, Schlafen 12 and/or CREB3L1, and radioimmunoassay. Elevated levels of PDE3A and Schlafen 12 polynucleotides or polypeptides relative to a reference are considered a positive indicator of cancer that is responsive to treatment with a PDE3A modulator. Reduced levels of a CREB3L1 or SLFN12 polynucleotide or polypeptide are considered an indicator of cancer that is resistant to treatment with a PDE3A modulator.

Types of Biological Samples

In characterizing the responsiveness of a malignancy in a subject to PDE3A modulator treatment, the level of PDE3A, SLFN12 and/or CREB3L1 expression is measured in different types of biologic samples. In one embodiment, the biologic sample is a tumor sample.

PDE3A and/or SLFN12 expression is higher in a sample obtained from a subject that is responsive to PDE3A modulator treatment than the level of expression in a non-responsive subject. In another embodiment, PDE3A and/or SLFN12 is at least about 5, 10, 20, or 30-fold higher in a subject with a malignancy than in a healthy control. Fold change values are determined using any method known in the art. In one embodiment, CREB3L1 or SLFN12 expression is reduced or undectable relative to a reference. In particular embodiments, CREB3L1 or SLFN12 expression is reduced by about 10%, 25%, 50%, 75%, 85%, 95% or more. In one embodiment, change is determined by calculating the difference in expression of PDE3A, SLFN12 and/or CREB3L1 in a cancer cell vs the level present in a non-responsive cancer cell or the level present in a corresponding healthy control cell.

Selection of a Treatment Method

As reported herein below, subjects suffering from a hyperproliferative disease may be tested for PDE3A, SLFN12 and/or CREB3L1 expression in the course of selecting a treatment method. Patients characterized as having increased PDE3A and/or SLFN12 relative to a reference level are identified as responsive to PDE3A modulator treatment. Subjects having reduced or undetectable levels of SLFN12 or CREB3L1 expression relative to a reference are identified as resistant to PDE3A modulator treatment.

Kits

The invention provides kits for characterizing the responsiveness or resistance of a subject to PDE3A modulator treatment.

Also provided herein are kits that can include a therapeutic composition containing an effective amount of a PDE3A modulator in, e.g., unit dosage form.

In one embodiment, a diagnostic kit of the invention provides a reagent for measuring relative expression of PDE3A and SLFN12. Such reagents include capture molecules (e.g., antibodies that recognize PDE3A and SLFN12 polypeptides or nucleic acid probes that hybridize with PDE3A and SLFN12 polynucleotides).

In another embodiment, a diagnostic kit includes a capture reagent (e.g., antibodies or nucleic acid probes) that binds CREB3L1 polypeptide or polynucleotide.

In some embodiments, the kit comprises a sterile container which includes a therapeutic or diagnostic composition; such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.

In one embodiment, a kit of the invention comprises reagents for measuring PDE3A, SLFN12 and/or CREB3L1 levels. If desired, the kit further comprises instructions for measuring PDE3A and/or SLFN12 and/or instructions for administering the PDE3A modulator to a subject having a malignancy, e.g., a malignancy selected as responsive to PDE3A modulator treatment. In particular embodiments, the instructions include at least one of the following: description of the therapeutic agent; dosage schedule and administration for treatment or prevention of malignancy or symptoms thereof; precautions; warnings; indications; counter-indications; over dosage information; adverse reactions; animal pharmacology; clinical studies; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.

The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (Freshney, 1987); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of the invention.

EXAMPLES Example 1. Identification of a Cell-Selective Cytotoxic Small Molecule

To identify anti-cancer compounds with cell-selective cytotoxic activity, an unbiased chemical screen was performed in two lung adenocarcinoma cell lines, A549 and NCI-H1734, both of which harbor oncogenic KRAS mutations and truncating STK11 mutations, and which were TP53 wild type and mutant (R273L), respectively. 1,924 compounds were screened from the Molecular Libraries Small-Molecule Repository validation set in the A549 and NCI-H1734 cell lines at a single concentration of 10 μM in 384-well format in duplicate. As a proxy for cellular viability, ATP content was measured after 48 hours of compound treatment.

Three compounds showed a selective reduction in cell viability for the NCI-H1734 cell line compared to the A549 cell line, with an approximately 50% reduction in the NCI-H1734 cell line, which is >4 median absolute deviations from the median in the negative direction, compared to a minimal change of <1 median absolute deviations from the median in the A549 cell line (FIG. 1A). Retesting the three compounds in a dose-response analysis validated that one compound, 6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one, or DNMDP, was specifically toxic to the NCI-H1734 cell line (FIG. 2).

Testing of additional cell lines with DNMDP showed clear cell-selective cytotoxicity, with an EC₅₀ between 10 and 100 nM for two additional lung adenocarcinoma cell lines, NCI-H1563 and NCI-H2122, and for HeLa cervical carcinoma cells, but an EC₅₀ greater than 1 μM for A549, MCF7, and PC3 cells (FIG. 1B; FIG. 1C). Thus one aspect of the invention is the use of DNMDP for the treatment of lung adenocarcinoma and cervical cancer. Caspase activity was detected by a caspase-sensitive luciferase assay and by poly ADP ribose polymerase (PARP) cleavage in HeLa cells upon DNMDP treatment, indicating that sensitive cells undergo apoptosis after DNMDP exposure (FIGS. 17A-17B).

To characterize cellular sensitivity to DNMDP further, 766 genomically characterized cancer cell lines were screened for DMNDP sensitivity at concentrations ranging from 66.4 μM to 2 nM in 2-fold dilution steps for 72 hours (see Large-scale cell-line viability measurements described further below). From these cell lines, 22 cell lines were categorized as sensitive with a robust Z-score lower than −4, which represented multiple lineages including multiple melanoma cell lines, amongst others (Table 1).

Next, the DNMDP enantiomers were separated by chiral super-critical fluid (SCF) chromatography. One enantiomer was 500-fold more potent in HeLa cells than the other (FIGS. 1C and D). The (R)-enantiomer was synthesized from commercially available starting materials (FIG. 3). This synthesized enantiomer had similar activity to the more potent separated material and was identical by chiral SCF chromatography, confirming stereochemistry of the active enantiomer (FIGS. 4A-4C). Two (R)-des-nitro analogues of DNMDP were synthesized, both of which tested similarly to (R)-DNMDP (FIG. 3). FIGS. 4A-4C show super-critical fluid (SCF) chromatographs of 6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3 (2H)-one (DNMDP) (top to bottom: ES+, diode array, ES− traces). FIG. 4A shows Peak 1 (CRO separation); FIG. 4B shows Peak 2 (CRO separation); and FIG. 4C shows synthesized (R)-DNMDP (5:95 ratio peaks 1:2 by uv).

TABLE 1 Sensitivity data of 766 cancer cell lines treated with DNMDP Cell line Lineage DNMDP AUC Robust Z-score COV318 OVARY 0.095838 −6.863450362 IGR37 SKIN 0.41146 −6.532158389 JHUEM1 ENDOMETRIUM 0.53468 −6.402820773 HEL HAEMATOPOIETIC AND 0.57955 −6.355723071 LYMPHOID TISSUE CORL51 LUNG 0.59436 −6.340177786 HEL9217 HAEMATOPOIETIC AND 0.75005 −6.176758102 LYMPHOID TISSUE NCIH1563 LUNG 1.0887 −5.821294837 SKMEL3 SKIN 1.2215 −5.681901594 NCIH2122 LUNG 1.3105 −5.58848293 RVH421 SKIN 1.4556 −5.436179018 HUT78 HAEMATOPOIETIC AND 1.5307 −5.35735046 LYMPHOID TISSUE DKMG CENTRAL NERVOUS SYSTEM 1.7217 −5.156867709 GB1 CENTRAL NERVOUS SYSTEM 1.8269 −5.046444748 G292CLONEA141B1 BONE 1.9664 −4.900018865 HMCB SKIN 1.9762 −4.889732315 A2058 SKIN 2.0833 −4.777315024 NCIH1734 LUNG 2.2179 −4.636032415 NCIH196 LUNG 2.5263 −4.312320999 LI7 LIVER 2.5414 −4.296471315 JHOM1 OVARY 2.7006 −4.129367368 COLO741 COLON 2.7231 −4.10575029 HS578T BREAST 2.8012 −4.023772788 K029AX SKIN 2.9362 −3.88207032 MONOMAC1 HAEMATOPOIETIC AND 2.9692 −3.847431939 LYMPHOID TISSUE HT1197 URINARY TRACT 3.0929 −3.717590492 NCIH520 LUNG 3.1351 −3.67329535 CAL78 BONE 3.1711 −3.635508025 NCIH647 LUNG 3.2187 −3.585544785 CGTHW1 THYROID 3.4296 −3.36417404 NCIH1666 LUNG 3.6097 −3.175132451 L33 PANCREAS 3.625 −3.159072838 UACC62 SKIN 3.9116 −2.858243747 CAS1 CENTRAL NERVOUS SYSTEM 3.9993 −2.766189625 CAL51 BREAST 4.0017 −2.76367047 OSRC2 KIDNEY 4.326 −2.423269652 X8505C THYROID 4.3418 −2.406685215 SH4 SKIN 4.3672 −2.380024158 NCIH1395 LUNG 4.4473 −2.29594736 SNU503 LARGE INTESTINE 4.5692 −2.16799528 HS729 SOFT TISSUE 4.6518 −2.081294362 SW579 THYROID 4.697 −2.033850277 YH13 CENTRAL NERVOUS SYSTEM 4.7007 −2.029966579 DBTRG05MG CENTRAL NERVOUS SYSTEM 4.7415 −1.987140944 SEM HAEMATOPOIETIC AND 4.7433 −1.985251578 LYMPHOID TISSUE HS852T SKIN 4.7511 −1.977064324 SNU449 LIVER 4.752 −1.976119641 NCIH2286 LUNG 4.7782 −1.948618866 JHOS2 OVARY 4.8254 −1.899075485 BICR31 UPPER AERODIGESTIVE 4.8356 −1.888369076 TRACT IGR1 SKIN 4.8613 −1.861393125 JHUEM3 ENDOMETRIUM 4.93 −1.789282313 SNU387 LIVER 4.9639 −1.753699249 UMUC1 URINARY TRACT 4.9933 −1.7228396 X8305C THYROID 5.0004 −1.7153871 NCIH1915 LUNG 5.0031 −1.712553051 P31FUJ HAEMATOPOIETIC AND 5.0106 −1.704680691 LYMPHOID TISSUE COLO678 LARGE INTESTINE 5.0245 −1.690090585 EOL1 HAEMATOPOIETIC AND 5.0478 −1.665633789 LYMPHOID TISSUE KNS42 CENTRAL NERVOUS SYSTEM 5.0791 −1.632779809 SW1783 CENTRAL NERVOUS SYSTEM 5.1161 −1.593942837 HS940T SKIN 5.1573 −1.550697343 SNU685 ENDOMETRIUM 5.206 −1.499579489 BCPAP THYROID 5.2336 −1.470609207 COLO829 SKIN 5.2432 −1.460532587 DM3 PLEURA 5.2635 −1.439224734 OCUM1 STOMACH 5.2843 −1.417392058 M059K CENTRAL NERVOUS SYSTEM 5.3059 −1.394719663 MG63 BONE 5.3943 −1.301930788 NCIH2172 LUNG 5.4245 −1.270231421 CAOV3 OVARY 5.4646 −1.228140539 PEER HAEMATOPOIETIC AND 5.4754 −1.216804342 LYMPHOID TISSUE HS839T SKIN 5.5232 −1.166631172 CORL105 LUNG 5.5442 −1.144588566 SNU5 STOMACH 5.5498 −1.138710537 MFE296 ENDOMETRIUM 5.5618 −1.126114762 NCIH854 LUNG 5.576 −1.111209762 NCIH146 LUNG 5.5773 −1.10984522 NCIH2081 LUNG 5.5811 −1.105856558 COV644 OVARY 5.5849 −1.101867896 VCAP PROSTATE 5.5863 −1.100398388 BICR18 UPPER AERODIGESTIVE 5.6 −1.086018212 TRACT RH18 SOFT TISSUE 5.6283 −1.056313176 KPNYN AUTONOMIC GANGLIA 5.6717 −1.010758457 KPNSI9S AUTONOMIC GANGLIA 5.6827 −0.99921233 SKCO1 LARGE INTESTINE 5.688 −0.993649196 MV411 HAEMATOPOIETIC AND 5.6905 −0.991025076 LYMPHOID TISSUE COV362 OVARY 5.6913 −0.990185358 NCO2 HAEMATOPOIETIC AND 5.7088 −0.971816519 LYMPHOID TISSUE JHH4 LIVER 5.71 −0.970556942 NCIH2141 LUNG 5.7218 −0.958171096 LXF289 LUNG 5.734 −0.945365392 MEWO SKIN 5.738 −0.9411668 TE125T SOFT TISSUE 5.744 −0.934868913 SNU869 BILIARY TRACT 5.7543 −0.924057539 LNCAPCLONEFGC PROSTATE 5.7557 −0.922588032 NCIH2009 LUNG 5.7594 −0.918704335 SKNBE2 AUTONOMIC GANGLIA 5.7717 −0.905793666 IALM LUNG 5.775 −0.902329827 DU145 PROSTATE 5.7825 −0.894457468 HCC1419 BREAST 5.7835 −0.89340782 NALM6 HAEMATOPOIETIC AND 5.7872 −0.889524123 LYMPHOID TISSUE PECAPJ15 UPPER AERODIGESTIVE 5.789 −0.887634757 TRACT LU99 LUNG 5.8016 −0.874409193 LAMA84 HAEMATOPOIETIC AND 5.8201 −0.854990707 LYMPHOID TISSUE ONCODG1 OVARY 5.8296 −0.845019051 HS888T BONE 5.8353 −0.839036058 SKNSH AUTONOMIC GANGLIA 5.8424 −0.831583558 TUHR14TKB KIDNEY 5.8451 −0.828749509 PF382 HAEMATOPOIETIC AND 5.8519 −0.821611903 LYMPHOID TISSUE ALLSIL HAEMATOPOIETIC AND 5.8724 −0.800094121 LYMPHOID TISSUE KMS34 HAEMATOPOIETIC AND 5.8799 −0.792221762 LYMPHOID TISSUE BICR6 UPPER AERODIGESTIVE 5.8837 −0.788233099 TRACT GRANTA519 HAEMATOPOIETIC AND 5.8937 −0.77773662 LYMPHOID TISSUE OCIAML2 HAEMATOPOIETIC AND 5.8945 −0.776896902 LYMPHOID TISSUE SUIT2 PANCREAS 5.8956 −0.775742289 BT549 BREAST 5.9226 −0.747401796 KMS28BM HAEMATOPOIETIC AND 5.9369 −0.732391831 LYMPHOID TISSUE HCC1428 BREAST 5.9402 −0.728927992 HCC1500 BREAST 5.9451 −0.723784718 A549 LUNG 5.9509 −0.71769676 KCL22 HAEMATOPOIETIC AND 5.9598 −0.708354893 LYMPHOID TISSUE COLO679 SKIN 5.9634 −0.704576161 SKMEL5 SKIN 5.9639 −0.704051337 HCC1395 BREAST 5.9716 −0.695969048 NCIH1435 LUNG 5.9756 −0.691770456 LOUNH91 LUNG 5.9793 −0.687886759 RPMI8402 HAEMATOPOIETIC AND 5.9827 −0.684317956 LYMPHOID TISSUE COLO668 LUNG 5.9969 −0.669412956 SKLU1 LUNG 6.0109 −0.654717885 KMS12BM HAEMATOPOIETIC AND 6.0135 −0.6519888 LYMPHOID TISSUE SNU1272 KIDNEY 6.0226 −0.642437004 MOLM6 HAEMATOPOIETIC AND 6.0447 −0.619239786 LYMPHOID TISSUE EPLC272H LUNG 6.0469 −0.61693056 SCC4 UPPER AERODIGESTIVE 6.0502 −0.613466722 TRACT LMSU STOMACH 6.0528 −0.610737638 KMS20 HAEMATOPOIETIC AND 6.0542 −0.60926813 LYMPHOID TISSUE G402 SOFT TISSUE 6.0606 −0.602550384 KYSE410 OESOPHAGUS 6.0741 −0.588380137 L540 HAEMATOPOIETIC AND 6.0807 −0.581452461 LYMPHOID TISSUE MOLT13 HAEMATOPOIETIC AND 6.084 −0.577988623 LYMPHOID TISSUE L1236 HAEMATOPOIETIC AND 6.0853 −0.57662408 LYMPHOID TISSUE LP1 HAEMATOPOIETIC AND 6.1029 −0.558150277 LYMPHOID TISSUE SNU620 STOMACH 6.1039 −0.557100629 MALME3M SKIN 6.112 −0.548598481 GSU STOMACH 6.1172 −0.543140312 MCF7 BREAST 6.1256 −0.53432327 COLO800 SKIN 6.1272 −0.532643833 MKN7 STOMACH 6.1453 −0.513645206 SNU119 OVARY 6.1473 −0.51154591 U118MG CENTRAL NERVOUS SYSTEM 6.1481 −0.510706192 OCILY19 HAEMATOPOIETIC AND 6.1512 −0.507452283 LYMPHOID TISSUE RKN SOFT TISSUE 6.1579 −0.500419642 DV90 LUNG 6.1676 −0.490238057 NCIH1355 LUNG 6.171 −0.486669254 KMM1 HAEMATOPOIETIC AND 6.1723 −0.485304712 LYMPHOID TISSUE NCIH1184 LUNG 6.1776 −0.479741578 U937 HAEMATOPOIETIC AND 6.1777 −0.479636613 LYMPHOID TISSUE EJM HAEMATOPOIETIC AND 6.1782 −0.479111789 LYMPHOID TISSUE C32 SKIN 6.1786 −0.47869193 NCIH23 LUNG 6.1854 −0.471554324 RERFLCAD1 LUNG 6.1862 −0.470714606 T3M10 LUNG 6.1867 −0.470189782 U266B1 HAEMATOPOIETIC AND 6.1906 −0.466096155 LYMPHOID TISSUE CAL54 KIDNEY 6.1949 −0.461582669 DND41 HAEMATOPOIETIC AND 6.1979 −0.458433726 LYMPHOID TISSUE PC14 LUNG 6.2003 −0.455914571 KMS11 HAEMATOPOIETIC AND 6.2008 −0.455389747 LYMPHOID TISSUE DMS53 LUNG 6.2061 −0.449826613 SNU1214 UPPER AERODIGESTIVE 6.2071 −0.448776965 TRACT GOS3 CENTRAL NERVOUS SYSTEM 6.2076 −0.448252141 TE8 OESOPHAGUS 6.2119 −0.443738655 ECGI10 OESOPHAGUS 6.2151 −0.440379781 KO52 HAEMATOPOIETIC AND 6.2174 −0.437965591 LYMPHOID TISSUE NCIH1793 LUNG 6.2189 −0.436391119 NB4 HAEMATOPOIETIC AND 6.219 −0.436286155 LYMPHOID TISSUE NCIH1105 LUNG 6.2191 −0.43618119 OCILY10 HAEMATOPOIETIC AND 6.222 −0.433137211 LYMPHOID TISSUE NCIH69 LUNG 6.2243 −0.430723021 A673 BONE 6.2304 −0.424320168 HCC4006 LUNG 6.2335 −0.42106626 SCC9 UPPER AERODIGESTIVE 6.2351 −0.419386823 TRACT OAW28 OVARY 6.2381 −0.416237879 BXPC3 PANCREAS 6.2387 −0.415608091 ISTMES1 PLEURA 6.2389 −0.415398161 SKMM2 HAEMATOPOIETIC AND 6.2396 −0.414663408 LYMPHOID TISSUE NCIN87 STOMACH 6.24 −0.414243548 T98G CENTRAL NERVOUS SYSTEM 6.2412 −0.412983971 GP2D LARGE INTESTINE 6.2536 −0.399968337 FTC238 THYROID 6.2564 −0.397029323 KMS27 HAEMATOPOIETIC AND 6.2607 −0.392515837 LYMPHOID TISSUE SNU201 CENTRAL NERVOUS SYSTEM 6.2618 −0.391361224 BC3C URINARY TRACT 6.266 −0.386952703 RS411 HAEMATOPOIETIC AND 6.2689 −0.383908724 LYMPHOID TISSUE TALL1 HAEMATOPOIETIC AND 6.2742 −0.37834559 LYMPHOID TISSUE RT4 URINARY TRACT 6.2742 −0.37834559 SKOV3 OVARY 6.2773 −0.375091681 RERFLCAD2 LUNG 6.2783 −0.374042033 KHM1B HAEMATOPOIETIC AND 6.2859 −0.366064709 LYMPHOID TISSUE KASUMI2 HAEMATOPOIETIC AND 6.2904 −0.361341294 LYMPHOID TISSUE MOLT16 HAEMATOPOIETIC AND 6.2966 −0.354833477 LYMPHOID TISSUE NUDUL1 HAEMATOPOIETIC AND 6.2966 −0.354833477 LYMPHOID TISSUE KMS18 HAEMATOPOIETIC AND 6.2973 −0.354098723 LYMPHOID TISSUE MDAMB175VII BREAST 6.2981 −0.353259005 RMGI OVARY 6.3019 −0.349270343 KIJK HAEMATOPOIETIC AND 6.305 −0.346016434 LYMPHOID TISSUE OCIAML5 HAEMATOPOIETIC AND 6.3062 −0.344756857 LYMPHOID TISSUE KMRC20 KIDNEY 6.3063 −0.344651892 LU65 LUNG 6.3082 −0.342657561 JIMT1 BREAST 6.3087 −0.342132737 SNU8 OVARY 6.3089 −0.341922807 KALS1 CENTRAL NERVOUS SYSTEM 6.3098 −0.340978124 SCABER URINARY TRACT 6.322 −0.32817242 OVMANA OVARY 6.3268 −0.32313411 TUHR10TKB KIDNEY 6.3302 −0.319565307 SUPM2 HAEMATOPOIETIC AND 6.3314 −0.318305729 LYMPHOID TISSUE JMSU1 URINARY TRACT 6.3317 −0.317990835 NCIH446 LUNG 6.3331 −0.316521328 COV434 OVARY 6.3341 −0.31547168 HCC38 BREAST 6.3361 −0.313372384 KMRC2 KIDNEY 6.3393 −0.310013511 SNU478 BILIARY TRACT 6.3432 −0.305919884 SUDHL1 HAEMATOPOIETIC AND 6.3444 −0.304660306 LYMPHOID TISSUE CMLT1 HAEMATOPOIETIC AND 6.3494 −0.299412067 LYMPHOID TISSUE UACC257 SKIN 6.3508 −0.29794256 NCIH1339 LUNG 6.3509 −0.297837595 M07E HAEMATOPOIETIC AND 6.3511 −0.297627665 LYMPHOID TISSUE KMRC3 KIDNEY 6.3514 −0.297312771 NCIH1693 LUNG 6.3603 −0.287970905 MM1S HAEMATOPOIETIC AND 6.3604 −0.28786594 LYMPHOID TISSUE HCC1143 BREAST 6.3611 −0.287131186 KATOIII STOMACH 6.3642 −0.283877278 MDAMB453 BREAST 6.3691 −0.278734003 J82 URINARY TRACT 6.3718 −0.275899954 CAL27 UPPER AERODIGESTIVE 6.3725 −0.2751652 TRACT HS766T PANCREAS 6.3727 −0.274955271 HCT8 LARGE INTESTINE 6.3733 −0.274325482 NCIH1581 LUNG 6.3747 −0.272855975 REH HAEMATOPOIETIC AND 6.3759 −0.271596397 LYMPHOID TISSUE MPP89 PLEURA 6.3817 −0.265508439 SNU761 LIVER 6.3819 −0.26529851 RH30 SOFT TISSUE 6.3841 −0.262989284 KURAMOCHI OVARY 6.3842 −0.26288432 HS936T SKIN 6.385 −0.262044601 HCC15 LUNG 6.3861 −0.260889989 F36P HAEMATOPOIETIC AND 6.388 −0.258895657 LYMPHOID TISSUE PANC0504 PANCREAS 6.3894 −0.25742615 NOMO1 HAEMATOPOIETIC AND 6.3925 −0.254172242 LYMPHOID TISSUE SKUT1 SOFT TISSUE 6.3987 −0.247664425 CCK81 LARGE INTESTINE 6.4043 −0.241786397 NCIH211 LUNG 6.4058 −0.240211925 NH6 AUTONOMIC GANGLIA 6.4066 −0.239372206 BECKER CENTRAL NERVOUS SYSTEM 6.4161 −0.229400551 NCIH1869 LUNG 6.4177 −0.227721114 ASPC1 PANCREAS 6.4186 −0.226776431 VMCUB1 URINARY TRACT 6.4199 −0.225411889 SNU398 LIVER 6.4206 −0.224677136 THP1 HAEMATOPOIETIC AND 6.4214 −0.223837417 LYMPHOID TISSUE HS611T HAEMATOPOIETIC AND 6.4224 −0.222787769 LYMPHOID TISSUE ONS76 CENTRAL NERVOUS SYSTEM 6.4253 −0.21974379 LOVO LARGE INTESTINE 6.4266 −0.218379248 GMS10 CENTRAL NERVOUS SYSTEM 6.4313 −0.213445903 RKO LARGE INTESTINE 6.4316 −0.213131009 ZR7530 BREAST 6.4339 −0.210716818 FU97 STOMACH 6.4421 −0.202109705 OCILY3 HAEMATOPOIETIC AND 6.4442 −0.199905445 LYMPHOID TISSUE BV173 HAEMATOPOIETIC AND 6.4448 −0.199275656 LYMPHOID TISSUE NCIH1568 LUNG 6.4489 −0.1949721 NCIH1155 LUNG 6.4497 −0.194132381 JURKAT HAEMATOPOIETIC AND 6.4524 −0.191298332 LYMPHOID TISSUE CW2 LARGE INTESTINE 6.4567 −0.186784846 RD SOFT TISSUE 6.4567 −0.186784846 RERFLCAI LUNG 6.4571 −0.186364987 YD10B UPPER AERODIGESTIVE 6.4579 −0.185525268 TRACT SF295 CENTRAL NERVOUS SYSTEM 6.4581 −0.185315339 JJN3 HAEMATOPOIETIC AND 6.4585 −0.18489548 LYMPHOID TISSUE EB1 HAEMATOPOIETIC AND 6.4633 −0.17985717 LYMPHOID TISSUE KNS60 CENTRAL NERVOUS SYSTEM 6.4642 −0.178912487 X697 HAEMATOPOIETIC AND 6.4674 −0.175553613 LYMPHOID TISSUE TOV21G OVARY 6.4695 −0.173349353 JHH5 LIVER 6.4703 −0.172509634 OVTOKO OVARY 6.4718 −0.170935162 WM1799 SKIN 6.4744 −0.168206078 PL21 HAEMATOPOIETIC AND 6.4754 −0.16715643 LYMPHOID TISSUE CA46 HAEMATOPOIETIC AND 6.4772 −0.165267064 LYMPHOID TISSUE PATU8988S PANCREAS 6.479 −0.163377697 HCC44 LUNG 6.4794 −0.162957838 KARPAS299 HAEMATOPOIETIC AND 6.4827 −0.159494 LYMPHOID TISSUE PANC0327 PANCREAS 6.4856 −0.156450021 YD8 UPPER AERODIGESTIVE 6.4856 −0.156450021 TRACT GDM1 HAEMATOPOIETIC AND 6.4875 −0.15445569 LYMPHOID TISSUE IM95 STOMACH 6.4877 −0.154245761 HCT15 LARGE INTESTINE 6.4918 −0.149942204 WM793 SKIN 6.4939 −0.147737944 SHP77 LUNG 6.5008 −0.140495373 X8MGBA CENTRAL NERVOUS SYSTEM 6.5012 −0.140075514 OUMS23 LARGE INTESTINE 6.5015 −0.139760619 SW1116 LARGE INTESTINE 6.5032 −0.137976218 NCIH1703 LUNG 6.5035 −0.137661324 HLF LIVER 6.5042 −0.13692657 REC1 HAEMATOPOIETIC AND 6.5051 −0.135981887 LYMPHOID TISSUE ML1 THYROID 6.5066 −0.134407415 HOS BONE 6.5069 −0.134092521 SW837 LARGE INTESTINE 6.5072 −0.133777626 EHEB HAEMATOPOIETIC AND 6.5124 −0.128319457 LYMPHOID TISSUE HUH28 BILIARY TRACT 6.5145 −0.126115197 MDAMB157 BREAST 6.5173 −0.123176182 CHP212 AUTONOMIC GANGLIA 6.5178 −0.122651359 RMUGS OVARY 6.52 −0.120342133 NCIH2106 LUNG 6.5249 −0.115198858 SKLMS1 SOFT TISSUE 6.5254 −0.114674034 X647V URINARY TRACT 6.5257 −0.11435914 HS294T SKIN 6.5258 −0.114254175 CHAGOK1 LUNG 6.5292 −0.110685372 NCIH2228 LUNG 6.5304 −0.109425795 MHHCALL3 HAEMATOPOIETIC AND 6.5324 −0.107326499 LYMPHOID TISSUE TE6 OESOPHAGUS 6.5328 −0.10690664 MHHES1 BONE 6.5353 −0.10428252 X42MGBA CENTRAL NERVOUS SYSTEM 6.5397 −0.099664069 SH10TC STOMACH 6.5448 −0.094310865 HCC202 BREAST 6.5484 −0.090532132 ACHN KIDNEY 6.5518 −0.08696333 SCC25 UPPER AERODIGESTIVE 6.5527 −0.086018646 TRACT PANC0403 PANCREAS 6.5578 −0.080665442 A2780 OVARY 6.5613 −0.076991674 EBC1 LUNG 6.5617 −0.076571815 SW620 LARGE INTESTINE 6.5658 −0.072268259 SKMEL31 SKIN 6.5659 −0.072163294 PK45H PANCREAS 6.5666 −0.07142854 NCIH2030 LUNG 6.5688 −0.069119315 SKMES1 LUNG 6.5724 −0.065340583 NAMALWA HAEMATOPOIETIC AND 6.5738 −0.063871075 LYMPHOID TISSUE CAL12T LUNG 6.5741 −0.063556181 HPBALL HAEMATOPOIETIC AND 6.5743 −0.063346251 LYMPHOID TISSUE HT1080 SOFT TISSUE 6.5745 −0.063136322 OE33 OESOPHAGUS 6.5749 −0.062716463 SR786 HAEMATOPOIETIC AND 6.5751 −0.062506533 LYMPHOID TISSUE NCIH929 HAEMATOPOIETIC AND 6.5755 −0.062086674 LYMPHOID TISSUE OVCAR4 OVARY 6.5755 −0.062086674 T47D BREAST 6.5764 −0.061141991 HCC1937 BREAST 6.5773 −0.060197308 SKHEP1 LIVER 6.5773 −0.060197308 KMS26 HAEMATOPOIETIC AND 6.5778 −0.059672484 LYMPHOID TISSUE SNU1066 UPPER AERODIGESTIVE 6.5779 −0.059567519 TRACT SUPHD1 HAEMATOPOIETIC AND 6.5802 −0.057153329 LYMPHOID TISSUE L428 HAEMATOPOIETIC AND 6.5828 −0.054424244 LYMPHOID TISSUE PLCPRF5 LIVER 6.584 −0.053164667 MSTO211H PLEURA 6.5871 −0.049910758 GA10 HAEMATOPOIETIC AND 6.59 −0.046866779 LYMPHOID TISSUE HSC2 UPPER AERODIGESTIVE 6.59 −0.046866779 TRACT MKN74 STOMACH 6.5911 −0.045712167 TOLEDO HAEMATOPOIETIC AND 6.5926 −0.044137695 LYMPHOID TISSUE KARPAS620 HAEMATOPOIETIC AND 6.5931 −0.043612871 LYMPHOID TISSUE CALU6 LUNG 6.5932 −0.043507906 SNU1196 BILIARY TRACT 6.5947 −0.041933434 HGC27 STOMACH 6.595 −0.04161854 NCIH716 LARGE INTESTINE 6.5964 −0.040149033 HDMYZ HAEMATOPOIETIC AND 6.5974 −0.039099385 LYMPHOID TISSUE A3KAW HAEMATOPOIETIC AND 6.6031 −0.033116392 LYMPHOID TISSUE SNGM ENDOMETRIUM 6.6038 −0.032381638 CAL851 BREAST 6.6074 −0.028602906 JHUEM2 ENDOMETRIUM 6.608 −0.027973117 LN18 CENTRAL NERVOUS SYSTEM 6.6106 −0.025244032 VMRCRCZ KIDNEY 6.6107 −0.025139067 TE10 OESOPHAGUS 6.6127 −0.023039772 CAKI2 KIDNEY 6.614 −0.021675229 PK1 PANCREAS 6.6156 −0.019995793 TE1 OESOPHAGUS 6.6158 −0.019785863 IGR39 SKIN 6.6163 −0.019261039 NCIH1781 LUNG 6.6169 −0.01863125 A253 SALIVARY GLAND 6.6238 −0.01138868 NCIH727 LUNG 6.6253 −0.009814208 G361 SKIN 6.6284 −0.006560299 TYKNU OVARY 6.6296 −0.005300722 SNU1041 UPPER AERODIGESTIVE 6.6307 −0.004146109 TRACT JL1 PLEURA 6.6309 −0.00393618 SNU283 LARGE INTESTINE 6.6315 −0.003306391 HCT116 LARGE INTESTINE 6.632 −0.002781567 LS1034 LARGE INTESTINE 6.6323 −0.002466673 EFO21 OVARY 6.633 −0.001731919 DMS114 LUNG 6.6335 −0.001207095 SNU1077 ENDOMETRIUM 6.6342 −0.000472342 DAOY CENTRAL NERVOUS SYSTEM 6.6343 −0.000367377 NCIH2342 LUNG 6.6346 −5.24824E−05 MOLP8 HAEMATOPOIETIC AND 6.6347  5.24824E−05 LYMPHOID TISSUE BHT101 THYROID 6.6351 0.000472342 TE5 OESOPHAGUS 6.6355 0.000892201 PSN1 PANCREAS 6.6403 0.005930511 NCIH2170 LUNG 6.6424 0.008134771 RCHACV HAEMATOPOIETIC AND 6.6426 0.008344701 LYMPHOID TISSUE HUH6 LIVER 6.6437 0.009499314 NCIH838 LUNG 6.6448 0.010653926 YAPC PANCREAS 6.6485 0.014537624 KYSE450 OESOPHAGUS 6.6505 0.016636919 RERFLCMS LUNG 6.6512 0.017371673 OVISE OVARY 6.6514 0.017581603 HT55 LARGE INTESTINE 6.6554 0.021780194 SNU899 UPPER AERODIGESTIVE 6.662 0.02870787 TRACT NCIH226 LUNG 6.6624 0.02912773 X639V URINARY TRACT 6.6635 0.030282342 TE14 OESOPHAGUS 6.6652 0.032066744 MKN45 STOMACH 6.6662 0.033116392 UMUC3 URINARY TRACT 6.6662 0.033116392 HEC6 ENDOMETRIUM 6.6667 0.033641216 X253JBV URINARY TRACT 6.6694 0.036475265 SKMEL24 SKIN 6.6712 0.038364631 VMRCLCD LUNG 6.6718 0.03899442 DLD1 LARGE INTESTINE 6.6751 0.042458258 ECC12 STOMACH 6.6785 0.046027061 WSUDLCL2 HAEMATOPOIETIC AND 6.6801 0.047706498 LYMPHOID TISSUE PFEIFFER HAEMATOPOIETIC AND 6.6804 0.048021392 LYMPHOID TISSUE NCIH2087 LUNG 6.6806 0.048231322 NCIH2029 LUNG 6.6826 0.050330617 SJSA1 BONE 6.6844 0.052219984 A172 CENTRAL NERVOUS SYSTEM 6.6858 0.053689491 SNU1033 LARGE INTESTINE 6.6873 0.055263963 TM31 CENTRAL NERVOUS SYSTEM 6.6885 0.05652354 X2313287 STOMACH 6.6886 0.056628505 SQ1 LUNG 6.6945 0.062821428 SUPT11 HAEMATOPOIETIC AND 6.695 0.063346251 LYMPHOID TISSUE NCIH2023 LUNG 6.6954 0.063766111 HCC1569 BREAST 6.6976 0.066075336 TT2609C02 THYROID 6.7014 0.070063998 SW1990 PANCREAS 6.7019 0.070588822 OVSAHO OVARY 6.7028 0.071533505 NCIH841 LUNG 6.7036 0.072373224 ME1 HAEMATOPOIETIC AND 6.7039 0.072688118 LYMPHOID TISSUE COLO205 LARGE INTESTINE 6.7052 0.07405266 TCCSUP URINARY TRACT 6.7056 0.074472519 TE11 OESOPHAGUS 6.7063 0.075207273 TE4 OESOPHAGUS 6.707 0.075942026 NCIH1694 LUNG 6.7095 0.078566146 KP4 PANCREAS 6.7102 0.0793009 CL11 LARGE INTESTINE 6.711 0.080140618 NCIH596 LUNG 6.7123 0.08150516 OCIAML3 HAEMATOPOIETIC AND 6.7152 0.084549139 LYMPHOID TISSUE KMH2 HAEMATOPOIETIC AND 6.7155 0.084864034 LYMPHOID TISSUE PK59 PANCREAS 6.7163 0.085703752 HDLM2 HAEMATOPOIETIC AND 6.7172 0.086648435 LYMPHOID TISSUE ES2 OVARY 6.7183 0.087803048 SKNDZ AUTONOMIC GANGLIA 6.7192 0.088747731 NCIH650 LUNG 6.7194 0.088957661 CAL62 THYROID 6.721 0.090637097 MDAMB231 BREAST 6.7222 0.091896675 HARA LUNG 6.7238 0.093576111 MFE319 ENDOMETRIUM 6.7242 0.093995971 LCLC103H LUNG 6.7269 0.09683002 OE19 OESOPHAGUS 6.7273 0.097249879 HT144 SKIN 6.7297 0.099769034 HEC251 ENDOMETRIUM 6.7301 0.100188893 A4FUK HAEMATOPOIETIC AND 6.7317 0.10186833 LYMPHOID TISSUE K562 HAEMATOPOIETIC AND 6.7319 0.102078259 LYMPHOID TISSUE HEC59 ENDOMETRIUM 6.7321 0.102288189 NCIH1341 LUNG 6.7337 0.103967626 A204 SOFT TISSUE 6.7338 0.10407259 OV7 OVARY 6.7346 0.104912309 OV90 OVARY 6.7381 0.108586076 HCC827 LUNG 6.7384 0.108900971 DU4475 BREAST 6.742 0.112679703 SKMEL1 SKIN 6.742 0.112679703 KYSE70 OESOPHAGUS 6.7428 0.113519422 CHP126 AUTONOMIC GANGLIA 6.7459 0.11677333 DETROIT562 UPPER AERODIGESTIVE 6.7465 0.117403119 TRACT CMK HAEMATOPOIETIC AND 6.7483 0.119292485 LYMPHOID TISSUE X769P KIDNEY 6.7486 0.11960738 DEL HAEMATOPOIETIC AND 6.7494 0.120447098 LYMPHOID TISSUE PANC0813 PANCREAS 6.751 0.122126535 COLO201 LARGE INTESTINE 6.752 0.123176182 SKNMC BONE 6.7533 0.124540725 CALU3 LUNG 6.7536 0.124855619 SNU1076 UPPER AERODIGESTIVE 6.7574 0.128844281 TRACT HCC78 LUNG 6.7625 0.134197486 ESS1 ENDOMETRIUM 6.7626 0.13430245 NCIH1755 LUNG 6.771 0.143119493 HPAFII PANCREAS 6.7751 0.147423049 CAKI1 KIDNEY 6.7755 0.147842908 COLO783 SKIN 6.778 0.150467028 NCIH2405 LUNG 6.7785 0.150991852 KNS81 CENTRAL NERVOUS SYSTEM 6.7793 0.15183157 HCC95 LUNG 6.7794 0.151936535 HL60 HAEMATOPOIETIC AND 6.7796 0.152146465 LYMPHOID TISSUE FADU UPPER AERODIGESTIVE 6.7809 0.153511007 TRACT TE617T SOFT TISSUE 6.782 0.15466562 KMBC2 URINARY TRACT 6.7837 0.156450021 HCC1171 LUNG 6.7838 0.156554986 CAPAN1 PANCREAS 6.786 0.158864211 CORL88 LUNG 6.7915 0.164637275 PECAPJ49 UPPER AERODIGESTIVE 6.7927 0.165896852 TRACT SF126 CENTRAL NERVOUS SYSTEM 6.7933 0.166526641 GSS STOMACH 6.794 0.167261395 U87MG CENTRAL NERVOUS SYSTEM 6.7949 0.168206078 HEYA8 OVARY 6.7972 0.170620268 HT1376 URINARY TRACT 6.7994 0.172929493 COLO792 SKIN 6.7997 0.173244388 SKMEL2 SKIN 6.8019 0.175553613 NCIH460 LUNG 6.8048 0.178597592 KU1919 URINARY TRACT 6.8061 0.179962134 SNU407 LARGE INTESTINE 6.8062 0.180067099 KU812 HAEMATOPOIETIC AND 6.8063 0.180172064 LYMPHOID TISSUE NCIH747 LARGE INTESTINE 6.8075 0.181431642 A101D SKIN 6.8089 0.182901149 PATU8988T PANCREAS 6.8099 0.183950797 HS895T SKIN 6.8118 0.185945128 HMC18 BREAST 6.8147 0.188989107 X253J URINARY TRACT 6.8153 0.189618895 TE9 OESOPHAGUS 6.8154 0.18972386 LS123 LARGE INTESTINE 6.8175 0.191928121 MCAS OVARY 6.8199 0.194447276 SW403 LARGE INTESTINE 6.8208 0.195391959 MDST8 LARGE INTESTINE 6.8209 0.195496924 RCM1 LARGE INTESTINE 6.8231 0.197806149 NCIH1650 LUNG 6.825 0.19980048 RPMI8226 HAEMATOPOIETIC AND 6.8256 0.200430269 LYMPHOID TISSUE SUDHL8 HAEMATOPOIETIC AND 6.8258 0.200640198 LYMPHOID TISSUE HEPG2 LIVER 6.8274 0.202319635 HT115 LARGE INTESTINE 6.8303 0.205363614 KYSE520 OESOPHAGUS 6.8305 0.205573544 ISHIKAWAHERAKLIO02ER ENDOMETRIUM 6.8313 0.206413262 RT112 URINARY TRACT 6.8313 0.206413262 SNU308 BILIARY TRACT 6.8314 0.206518227 HCC1806 BREAST 6.8314 0.206518227 NCIH2085 LUNG 6.8317 0.206833121 EFO27 OVARY 6.832 0.207148015 NCIH2052 PLEURA 6.8321 0.20725298 HSC4 UPPER AERODIGESTIVE 6.8327 0.207882769 TRACT KYSE140 OESOPHAGUS 6.836 0.211346607 LC1SQSF LUNG 6.8361 0.211451572 KMRC1 KIDNEY 6.8362 0.211556537 HUPT3 PANCREAS 6.837 0.212396255 NCIH1838 LUNG 6.8375 0.212921079 T24 URINARY TRACT 6.8383 0.213760797 WM115 SKIN 6.8396 0.21512534 KASUMI1 HAEMATOPOIETIC AND 6.8439 0.219638826 LYMPHOID TISSUE GAMG CENTRAL NERVOUS SYSTEM 6.8471 0.222997699 SBC5 LUNG 6.8494 0.225411889 WM2664 SKIN 6.8521 0.228245938 D283MED CENTRAL NERVOUS SYSTEM 6.857 0.233389213 MIAPACA2 PANCREAS 6.8607 0.23727291 BL70 HAEMATOPOIETIC AND 6.8619 0.238532488 LYMPHOID TISSUE NCIH1623 LUNG 6.862 0.238637453 BHY UPPER AERODIGESTIVE 6.8627 0.239372206 TRACT OVCAR8 OVARY 6.8637 0.240421854 SNU840 OVARY 6.8651 0.241891361 CFPAC1 PANCREAS 6.8671 0.243990657 HS944T SKIN 6.8697 0.246719742 LK2 LUNG 6.8724 0.249553791 JHH1 LIVER 6.8737 0.250918333 OVKATE OVARY 6.8742 0.251443157 T84 LARGE INTESTINE 6.8791 0.256586432 SW1573 LUNG 6.8813 0.258895657 KYSE30 OESOPHAGUS 6.8825 0.260155235 DANG PANCREAS 6.8825 0.260155235 SU8686 PANCREAS 6.8851 0.26288432 YD15 SALIVARY GLAND 6.8858 0.263619073 COLO680N OESOPHAGUS 6.8864 0.264248862 SUDHL6 HAEMATOPOIETIC AND 6.887 0.264878651 LYMPHOID TISSUE SNU626 CENTRAL NERVOUS SYSTEM 6.8886 0.266558087 SNU1105 CENTRAL NERVOUS SYSTEM 6.8918 0.269916961 BT20 BREAST 6.8931 0.271281503 FTC133 THYROID 6.8949 0.273170869 P12ICHIKAWA HAEMATOPOIETIC AND 6.8951 0.273380799 LYMPHOID TISSUE NCIH292 LUNG 6.8954 0.273695693 JHH2 LIVER 6.9004 0.278943933 RCC10RGB KIDNEY 6.9009 0.279468757 JHOC5 OVARY 6.9036 0.282302806 X786O KIDNEY 6.9057 0.284507067 AN3CA ENDOMETRIUM 6.9081 0.287026222 KP3 PANCREAS 6.909 0.287970905 HEC151 ENDOMETRIUM 6.9099 0.288915588 KE39 STOMACH 6.9103 0.289335447 HS822T BONE 6.9115 0.290595024 A375 SKIN 6.9117 0.290804954 MORCPR LUNG 6.9126 0.291749637 C2BBE1 LARGE INTESTINE 6.9144 0.293639003 NCIH2452 PLEURA 6.9169 0.296263123 TCCPAN2 PANCREAS 6.9184 0.297837595 VMRCRCW KIDNEY 6.9222 0.301826257 NCIH810 LUNG 6.9222 0.301826257 PC3 PROSTATE 6.9226 0.302246116 MDAMB435S SKIN 6.9227 0.302351081 NCIH322 LUNG 6.9254 0.30518513 MOLP2 HAEMATOPOIETIC AND 6.928 0.307914215 LYMPHOID TISSUE HCC366 LUNG 6.9295 0.309488687 KELLY AUTONOMIC GANGLIA 6.9352 0.31547168 AGS STOMACH 6.9378 0.318200764 MDAMB468 BREAST 6.9388 0.319250412 SNUC5 LARGE INTESTINE 6.939 0.319460342 HCC1195 LUNG 6.941 0.321559638 NB1 AUTONOMIC GANGLIA 6.9466 0.327437666 NCIH2126 LUNG 6.9473 0.32817242 HT HAEMATOPOIETIC AND 6.9476 0.328487314 LYMPHOID TISSUE SW48 LARGE INTESTINE 6.9505 0.331531293 QGP1 PANCREAS 6.9517 0.33279087 NUGC3 STOMACH 6.9527 0.333840518 SNU719 STOMACH 6.9544 0.33562492 SKES1 BONE 6.9576 0.338983793 OVK18 OVARY 6.9579 0.339298688 HEC1B ENDOMETRIUM 6.9583 0.339718547 KLE ENDOMETRIUM 6.9584 0.339823511 HEC50B ENDOMETRIUM 6.9622 0.343812174 TF1 HAEMATOPOIETIC AND 6.9682 0.350110061 LYMPHOID TISSUE AM38 CENTRAL NERVOUS SYSTEM 6.9715 0.353573899 HCC1954 BREAST 6.9728 0.354938441 MELHO SKIN 6.9769 0.359241998 EN ENDOMETRIUM 6.9773 0.359661857 HCC2108 LUNG 6.9789 0.361341294 X22RV1 PROSTATE 6.9813 0.363860449 PATU8902 PANCREAS 6.9874 0.370263301 LN229 CENTRAL NERVOUS SYSTEM 6.9883 0.371207984 GI1 CENTRAL NERVOUS SYSTEM 6.9897 0.372677491 SNU213 PANCREAS 6.9923 0.375406576 COLO684 ENDOMETRIUM 6.993 0.376141329 SNU738 CENTRAL NERVOUS SYSTEM 6.9945 0.377715801 JK1 HAEMATOPOIETIC AND 6.9966 0.379920062 LYMPHOID TISSUE KYSE510 OESOPHAGUS 6.9987 0.382124322 NCIH1299 LUNG 6.9991 0.382544181 IGROV1 OVARY 7.0026 0.386217949 ACCMESO1 PLEURA 7.0033 0.386952703 BICR16 UPPER AERODIGESTIVE 7.0071 0.390941365 TRACT HCC2279 LUNG 7.0072 0.39104633 PANC1 PANCREAS 7.0096 0.393565485 CCFSTTG1 CENTRAL NERVOUS SYSTEM 7.0119 0.395979675 SNU668 STOMACH 7.0126 0.396714428 SW1271 LUNG 7.0143 0.39849883 SUDHL4 HAEMATOPOIETIC AND 7.0162 0.400493161 LYMPHOID TISSUE GCT SOFT TISSUE 7.0174 0.401752738 TT THYROID 7.0183 0.402697421 DMS454 LUNG 7.019 0.403432175 LS180 LARGE INTESTINE 7.0225 0.407105943 SNU182 LIVER 7.0252 0.409939992 KNS62 LUNG 7.0253 0.410044957 OC314 OVARY 7.0273 0.412144253 RH41 SOFT TISSUE 7.0285 0.41340383 NCIH1373 LUNG 7.0318 0.416867668 BEN LUNG 7.0341 0.419281858 MESSA SOFT TISSUE 7.0401 0.425579746 HEC1A ENDOMETRIUM 7.0465 0.432297493 L363 HAEMATOPOIETIC AND 7.0473 0.433137211 LYMPHOID TISSUE CAL29 URINARY TRACT 7.0497 0.435656366 RAJI HAEMATOPOIETIC AND 7.0524 0.438490415 LYMPHOID TISSUE ZR751 BREAST 7.054 0.440169852 KYSE180 OESOPHAGUS 7.0541 0.440274817 LOXIMVI SKIN 7.058 0.444368444 YD38 UPPER AERODIGESTIVE 7.06 0.446467739 TRACT SNU410 PANCREAS 7.0646 0.45129612 NCIH2291 LUNG 7.0654 0.452135838 PANC0203 PANCREAS 7.0662 0.452975556 NCIH1792 LUNG 7.0701 0.457069183 SW1088 CENTRAL NERVOUS SYSTEM 7.0786 0.46599119 SKMEL30 SKIN 7.079 0.46641105 KM12 LARGE INTESTINE 7.0792 0.466620979 HEC108 ENDOMETRIUM 7.0804 0.467880557 NCIH526 LUNG 7.0825 0.470084817 NCIH661 LUNG 7.0832 0.470819571 KYSE150 OESOPHAGUS 7.0859 0.47365362 TUHR4TKB KIDNEY 7.0861 0.47386355 U251MG CENTRAL NERVOUS SYSTEM 7.091 0.479006825 MKN1 STOMACH 7.0915 0.479531649 DMS273 LUNG 7.0958 0.484045135 HS683 CENTRAL NERVOUS SYSTEM 7.0975 0.485829536 HS746T STOMACH 7.1012 0.489713233 OAW42 OVARY 7.1038 0.492442318 KYO1 HAEMATOPOIETIC AND 7.1048 0.493491966 LYMPHOID TISSUE HS688AT SKIN 7.1049 0.493596931 SIGM5 HAEMATOPOIETIC AND 7.1077 0.496535945 LYMPHOID TISSUE HUCCT1 BILIARY TRACT 7.1094 0.498320346 HS819T BONE 7.1097 0.498635241 HCC1588 LUNG 7.1149 0.50409341 KPL1 BREAST 7.1178 0.507137389 KE97 HAEMATOPOIETIC AND 7.1187 0.508082072 LYMPHOID TISSUE HCC2218 BREAST 7.1208 0.510286332 OCIM1 HAEMATOPOIETIC AND 7.1253 0.515009748 LYMPHOID TISSUE NCIH441 LUNG 7.1284 0.518263657 NCIH1092 LUNG 7.139 0.529389924 SKMEL28 SKIN 7.1392 0.529599854 HPAC PANCREAS 7.1394 0.529809784 SAOS2 BONE 7.1406 0.531069361 RL952 ENDOMETRIUM 7.1432 0.533798446 SKNAS AUTONOMIC GANGLIA 7.145 0.535687812 CAL148 BREAST 7.1477 0.538521861 DMS79 LUNG 7.1572 0.548493516 EFE184 ENDOMETRIUM 7.1614 0.552902038 SUPT1 HAEMATOPOIETIC AND 7.167 0.558780066 LYMPHOID TISSUE NMCG1 CENTRAL NERVOUS SYSTEM 7.1746 0.56675739 NCIH358 LUNG 7.1753 0.567492144 TE441T SOFT TISSUE 7.1772 0.569486475 MELJUSO SKIN 7.1877 0.580507778 IPC298 SKIN 7.1984 0.59173901 SW1353 BONE 7.1985 0.591843975 CAL33 UPPER AERODIGESTIVE 7.2038 0.597407109 TRACT SNU489 CENTRAL NERVOUS SYSTEM 7.2056 0.599296475 LCLC97TM1 LUNG 7.2086 0.602445419 BICR56 UPPER AERODIGESTIVE 7.2108 0.604754644 TRACT NCIH508 LARGE INTESTINE 7.2176 0.61189225 HSC3 UPPER AERODIGESTIVE 7.2237 0.618295103 TRACT SNU878 LIVER 7.2238 0.618400067 CAMA1 BREAST 7.2254 0.620079504 LS411N LARGE INTESTINE 7.2279 0.622703624 YKG1 CENTRAL NERVOUS SYSTEM 7.2376 0.632885208 JHH6 LIVER 7.2377 0.632990173 KG1C CENTRAL NERVOUS SYSTEM 7.238 0.633305068 BT474 BREAST 7.2422 0.637713589 SNU1079 BILIARY TRACT 7.2463 0.642017145 KARPAS422 HAEMATOPOIETIC AND 7.2487 0.6445363 LYMPHOID TISSUE HEC265 ENDOMETRIUM 7.2509 0.646845526 NCIH2444 LUNG 7.2606 0.65702711 NUDHL1 HAEMATOPOIETIC AND 7.2677 0.664479611 LYMPHOID TISSUE AMO1 HAEMATOPOIETIC AND 7.2764 0.673611547 LYMPHOID TISSUE HCC1833 LUNG 7.2887 0.686522217 SNUC4 LARGE INTESTINE 7.2927 0.690720808 HDQP1 BREAST 7.2935 0.691560527 OV56 OVARY 7.2957 0.693869752 P3HR1 HAEMATOPOIETIC AND 7.2973 0.695549189 LYMPHOID TISSUE NUGC4 STOMACH 7.2991 0.697438555 U2OS BONE 7.3013 0.69974778 SNU886 LIVER 7.3032 0.701742112 NCIH28 PLEURA 7.3081 0.706885386 SNU601 STOMACH 7.3091 0.707935034 ECC10 STOMACH 7.3182 0.71748683 LS513 LARGE INTESTINE 7.3199 0.719271232 CAL120 BREAST 7.32 0.719376196 SNU1040 LARGE INTESTINE 7.3288 0.728613098 NCIH2171 LUNG 7.3416 0.742048591 SUDHL5 HAEMATOPOIETIC AND 7.3508 0.751705352 LYMPHOID TISSUE BFTC905 URINARY TRACT 7.3514 0.752335141 HT29 LARGE INTESTINE 7.364 0.765560705 RPMI7951 SKIN 7.375 0.777106832 AML193 HAEMATOPOIETIC AND 7.3753 0.777421726 LYMPHOID TISSUE MEC1 HAEMATOPOIETIC AND 7.376 0.778156479 LYMPHOID TISSUE HEP3B217 LIVER 7.4062 0.809855846 SNU475 LIVER 7.4091 0.812899825 HUH1 LIVER 7.4298 0.834627537 HUPT4 PANCREAS 7.4555 0.861603488 IMR32 AUTONOMIC GANGLIA 7.4593 0.865592151 NCIH889 LUNG 7.4952 0.903274511 HCC2935 LUNG 7.5084 0.917129863 MC116 HAEMATOPOIETIC AND 7.5146 0.92363768 LYMPHOID TISSUE X5637 URINARY TRACT 7.5183 0.927521377 SKM1 HAEMATOPOIETIC AND 7.5234 0.932874582 LYMPHOID TISSUE SKBR3 BREAST 7.5494 0.960165427 EM2 HAEMATOPOIETIC AND 7.5755 0.987561238 LYMPHOID TISSUE RI1 HAEMATOPOIETIC AND 7.5915 1.004355605 LYMPHOID TISSUE SIMA AUTONOMIC GANGLIA 7.6032 1.016636485 FUOV1 OVARY 7.6122 1.026083316 SNUC2A LARGE INTESTINE 7.6165 1.030596802 SNU61 LARGE INTESTINE 7.6228 1.037209584 CAPAN2 PANCREAS 7.6273 1.041933 SNU216 STOMACH 7.6319 1.04676138 MOLM13 HAEMATOPOIETIC AND 7.646 1.061561416 LYMPHOID TISSUE HUNS1 HAEMATOPOIETIC AND 7.6648 1.081294796 LYMPHOID TISSUE HCC1438 LUNG 7.7264 1.145953108 NCIH2196 LUNG 7.7386 1.158758812 SNU466 CENTRAL NERVOUS SYSTEM 7.7589 1.180066665 SUDHL10 HAEMATOPOIETIC AND 7.7977 1.220793004 LYMPHOID TISSUE SNU46 UPPER AERODIGESTIVE 7.8035 1.226880962 TRACT CALU1 LUNG 7.8185 1.242625681 BFTC909 KIDNEY 7.9189 1.348010331 JVM3 HAEMATOPOIETIC AND 7.961 1.392200508 LYMPHOID TISSUE MHHCALL4 HAEMATOPOIETIC AND 8.031 1.465675862 LYMPHOID TISSUE JURLMK1 HAEMATOPOIETIC AND 8.1126 1.551327131 LYMPHOID TISSUE KE37 HAEMATOPOIETIC AND 8.1163 1.555210829 LYMPHOID TISSUE S117 SOFT TISSUE 8.2668 1.713182839 KMS21BM HAEMATOPOIETIC AND 8.3309 1.780465271 LYMPHOID TISSUE KYM1 SOFT TISSUE 8.4417 1.896766259 CORL95 LUNG 8.5762 2.037943903 MHHNB11 AUTONOMIC GANGLIA 8.8255 2.299621128 MDAMB361 BREAST 9.2909 2.788127266

Example 1b

Cell Proliferation Measurement

The antiproliferative activity of the compounds of the general formula (I) was examined in vitro in human cancer cells. For this purpose, 1000 cells were plated in 384-well plates with appropriate growth medium and incubated at 37° C. overnight. After 24 h, cells on one plate (0 h plate) were treated with 30 μl/cavity of CTG solution (Promega Cell Titer Glo (catalogue #G755B and G756B)) and incubated at room temperature for 10 min, and luminescence was measured by means of a VICTOR V (Perkin Elmer), in order to determine cell viability on commencement of treatment. The cells on the test plate were treated with the compounds of the general formula (I) as and incubated at 37° C. for 72 h. The compounds were added to the cells by means of an HP D300 digital dispenser in a 10-step 2,5-fold dilution series. As control, the cells were treated with vehicle (DMSO at 0.3% final concentration). After 72 h, the cells were treated with 30 μl/cavity of CTG solution (Promega Cell Titer Glo (catalogue #G755B and G756B)) and incubated at room temperature for 10 min, and luminescence was measured by means of a VICTOR V (Perkin Elmer), in order to determine cell viability at the end of treatment. The percentage effect on cell growth and the IC₅₀ derived therefrom were determined for each test substance using the values from the 0 h plate (=maximum inhibition) and the DMSO control (=minimum inhibition). The IC₅₀ values were calculated using a 4-parameter fit.

For compound 6 the following IC₅₀ were obtained:

TABLE 1a Cell proliferation results Cell line Indication IC50[M] A549 Lung adenocarcinoma >6.00E−7 (inactive) SKMEL3 Melanoma 4.41E−10 HeLa Cervical Cancer 3.27E−10

Thus another aspect of the invention is the use of compound 6 for the treatment of skin cancer (especially melanoma), and cervical cancer.

Example 2. Application of Predictive Chemogenomics and Identification of PDE3A as a Putative Target of DNMDP

Given the potent cell-selective growth inhibition by 6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one (DNMDP), its mechanism of action was examined in more detail. To determine the molecular target of DNMDP, chemogenomic analysis was performed of the 766 tested cell lines, previously characterized for mutations, copy number, and gene expression features as part of the Cancer Cell Line Encyclopedia (CCLE, Barretina et al., 2012), to look for correlation between these genomic features and DNMDP sensitivity. Analysis of Pearson correlations between DNMDP sensitivity and expression of individual genes across the cell line set showed a strong correlation with expression of the PDE3A gene, encoding phosphodiesterase 3A (FIG. 5A). The correlation between DNMDP sensitivity and PDE3A expression is not perfect (FIG. 18), and it is possible that some errors are introduced due to the high-throughput nature of the cell line sensitivity characterization, as manual validation for all 766 cell lines was not logistically feasible. Mutation and copy number features, in contrast, did not correlate with DNMDP sensitivity. Conversely, of 480 compounds tested, DNMDP sensitivity was the closest correlate of PDE3A expression (FIG. 5B), indicating that cancer cell lines with high PDE3A expression were more distinctly sensitive to DNMDP than to any other tested compound. In contrast to the motivation of the initial screen, there was no correlation between TP53 mutation, or other measures of p53 function, and DNMDP sensitivity.

Given these results and the clear structural similarity of DNMDP to known PDE3 inhibitors, e.g., levosimendan and siguazodan (FIGS. 6A-6C), biochemical analysis of DNMDP against 19 phosphodiesterases representing 11 PDE super families was performed. At a concentration of 100 nM, DNMDP specifically inhibited both PDE3A and PDE3B, weakly inhibited PDE10, and had little or no detectable effect on other phosphodiesterases (Table 2).

Because of the cellular correlation between PDE3A expression and DNMDP sensitivity, the in vitro inhibition of PDE3A and PDE3B by DNMDP, and the structural similarity of DNMDP to known PDE3 inhibitors, it was analyzed whether all PDE3 inhibitors would exhibit a similar cytotoxic profile to DNMDP. Surprisingly, there was almost no correlation between IC₅₀ for in vitro enzymatic PDE3A inhibition and HeLa cell cytotoxicity across a series of tested compounds (FIG. 5C and FIGS. 7A and 7B). Indeed, the potent PDE3 inhibitor trequinsin (PDE3 IC₅₀=0.25 nM, Ruppert et al., Life Sci. 31, 2037-2043, 1982) did not affect HeLa cell viability in any detectable way. Despite their differential effects on HeLa cell viability, the non-cytotoxic PDE3 inhibitor trequinsin and the potent cytotoxic compound DNMDP had similar effects on intracellular cAMP levels in forskolin-treated HeLa cells (FIGS. 8A and 8B). This result indicates that inhibition of the cAMP and cGMP hydrolysis functions of PDE3A was not sufficient for the cytotoxic activity of DNMDP.

TABLE 2 Results of phosphodiesterase inhibition reactions PDE % inh. #1 % inh. #2 % inhibition PDE1A1 3 7 5 PDE1B −5 0 −2 PDE1C 2 9 5 PDE2A 6 10 8 PDE3A 95 95 95 PDE3B 98 97 97 PDE4A1A 14 18 16 PDE4B1 21 20 21 PDE4C1 10 14 12 PDE4D3 14 16 15 PDE4D7 19 20 20 PDE5A1 16 16 16 PDE7A 24 20 22 PDE7B 5 11 8 PDE8A1 10 12 11 PDE9A2 0 5 2 PDE10A1 61 65 63 PDE10A2 67 70 68 PDE11A 14 18 16

Example 3. Target Validation of PDE3A

The complex relationship between phosphodiesterase 3A (PDE3A) inhibition and cell killing, in which 6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one (DNMDP) and some PDE3 inhibitors kill HeLa and other DNMDP-sensitive cells, whereas others PDE3 inhibitors do not affect cell viability, indicated several possible interpretations including: 1) the cytotoxic activity might be PDE3-independent and due to action on a different protein though screening 234 kinases found no kinase inhibition by 1 μM DNMDP; 2) cytotoxic and non-cytotoxic PDE3 inhibitors might bind to different sites within the protein and exert distinct activities; or 3) the cytotoxic and non-cytotoxic PDE3 inhibitors might bind to the PDE3 active sites but have different effects on the conformation and activity of the protein. This third possibility might be unexpected, but allosteric modulators of PDE4 have been shown to bind the PDE4 active site and interact with upstream (UCR2), and downstream (CR3) regulatory domains and thereby stabilize specific inactive conformations (Burgin et al., Nat Biotechnol 28, 63-70, 2010). Most importantly, PDE4 competitive inhibitors and PDE4 allosteric modulators with similar IC₅₀s for cAMP hydrolysis in vitro had different cellular activities and safety profiles in animal studies (Burgin et al., Nat Biotechnol 28, 63-70, 2010). To evaluate whether PDE inhibitors or other small molecules compete with DNMDP, the PHARMAKON 1600 collection of 1600 bioactive compounds (PHARMAKON 1600 is a unique collection of 1600 known drugs from US and International Pharmacopeia) was screened to identify compounds that were able to rescue cell death induced by DNMDP. HeLa cells were co-treated with nM DNMDP (the EC₇₀ concentration) and 2 μM of each bioactive compound. Cell viability after 48-hour treatment was assessed by ATP consumption as described earlier. The five most potent compounds that rescued cell death induced by DNMDP were all PDE inhibitors, and the three most potent compounds, levosimendan, milrinone, and cilostazol, were all selective PDE3 inhibitors (FIG. 9A).

In follow-up experiments, it was confirmed that cilostamide, levosimendan, milrinone, and several other non-cytotoxic selective PDE3 inhibitors were able to rescue DNMDP cytotoxicity in a dose-dependent manner (FIG. 9B). The most potent DNMDP competitor was trequinsin, with an “RC₅₀” (the concentration at which it achieved 50% rescue) of <1 nM; in contrast, PDE5 inhibitors such as sildenafil and vardenafil, as well as the pan-PDE inhibitors idubulast and dipyridamole, were not effective competitors up to 1 μM concentrations in this assay (FIG. 9B). This indicated that non-cytotoxic PDE3 inhibitors and DNMDP compete for binding to the same molecular target that is mediating the cytotoxic phenotype.

To identify the molecular target of DNMDP, an affinity purification was performed using an (R)-des-nitro-DNMDP solid-phase tethered linker analogue (FIG. 10A) incubated with HeLa cell lysate. This linker analogue had the same DNMDP cytotoxicity rescue phenotype as non-cytotoxic PDE3 inhibitors described above (FIG. 10B), indicating that it too bound to the same molecular target. It was competed for the molecular target by adding either an excess of trequinsin or separate enantiomers of DNMDP, where only the (R)-enantiomer was cytotoxic. Immunoblotting for PDE3A of the affinity purified material showed that PDE3A indeed binds to the linker analogue. Binding of PDE3A to the linker analogue was blocked by both trequinsin and (R)-DNMDP, but not by the non-cytotoxic enantiomer (S)-DNMDP (FIG. 9C). Thus both trequinsin and (R)-DNMDP prevented the binding of PDE3A to the tethered DNMDP analogue, and it was concluded that both molecules bind PDE3A directly.

Based on the observations that DNMDP-sensitive cells expressed high levels of PDE3A, and that DNMDP competed with non-cytotoxic inhibitors for PDE3A binding, it was hypothesized that DNMDP mediated its cytotoxic phenotype through the interaction with PDE3A and that PDE3A abundance was a direct cellular determinant of DNMDP sensitivity. To validate this hypothesis, the effect of reducing levels of PDE3A on the response to DNMDP was tested. A clustered regularly interspaced short palindromic (CRISPR)-associated CAS9 enzyme that was targeted with three guide RNAs (sgRNA) targeting three different sites in the PDE3A locus led to complete loss of PDE3A expression (Cong et al., Science 339, 819-823, 2013) sgRNA2 and sgRNA3 almost completely reduced PDE3A protein levels, whereas sgRNA1 had a moderate effect on PDE3A expression (FIG. 11A). Importantly, both sgRNA2 and sgRNA3 led to significant rescue of toxicity by an active cytotoxic DNMDP analog, 3 (FIGS. 11A and 11B and FIGS. 5A-5C). Both sgRNA2 and sgRNA3 led to significant rescue of toxicity by DNMDP (FIG. 11C). Changes in proliferation rate or morphology in HeLa cells with reduced PDE3A expression were not observed, indicating that PDE3A was not required for cell survival. In an independent approach using an siRNA smart-pool containing four different siRNAs targeting PDE3A, PDE3A expression was reduced in HeLa cell line with a maximum efficiency of 70% between 24 and 72 hours after transfection. HeLa cells treated with siPDE3A had a higher EC₅₀ to a DNMDP analog compared to the control siRNA condition (FIGS. 12A and 12B). Without being bound by theory it was concluded that DNMDP cytotoxicity requires PDE3A, and that DNMDP likely modulates the function of PDE3A.

Example 4. Determining the Mechanism of Action of DNMDP

The dependence of 6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one (DNMDP) cytotoxicity on phosphodiesterase 3A (PDE3A) protein abundance indicated a possible mechanism similar to that recently observed for lenalidomide, which acts by a neomorphic or hypermorphic mechanism by stabilizing an interaction between cereblon and IKAROS Family Zinc Finger 1 (IKZF1) and IKZF3 (Krönke et al., Science 343, 301-305, 2014; Lu et al., Science 343, 305-309, 2014). In addition, PDE4 allosteric modulators, but not competitive inhibitors, have been shown to bind and stabilize a “closed” protein conformation that has independently been shown to uniquely bind the PDE4-partner protein DISCi (Millar et al., Science 310, 1187-1191, 2005). The protein complexes in which PDE3A resides were characterized under normal conditions, and it was examined how these complexes change when PDE3A is bound to DNMDP or the non-cytotoxic PDE3 inhibitor trequinsin. PDE3A and interacting proteins from Hela cells were immunoprecipitated in the presence of DNMDP and trequinsin followed by labeling with isobaric stable isotope tags for relative abundance and quantitation by mass spectrometry (iTRAQ/MS, FIG. 13A). PDE3A immunoprecipitates from HeLa cells were enriched for multiple protein phosphatase subunits including protein phosphatase 2 subunits (PPP2CA, PPP2R1A, PPP2R1B, PPP2R2A, PPP2R2D), calcineurin (PPP3R1, PPP3CA, Beca et al., Circ. Res. 112, 289-297, 2013), 14-3-3 (YWHAB, YWHAQ, YWHAG, YWHAZ, Pozuelo Rubio et al., Biochem. J. 392, 163-172, 2005), and tubulin (TUBAIC, TUBAIB) family members (FIG. 13B and FIG. 14A). In addition, it was found that PDE3A and PDE3B reside in the same protein complex, which has been previously reported (Malovannaya et al., Cell 145, 787-799, 2011).

Binding of DNMDP altered the composition of interacting proteins that were co-immunoprecipitated with PDE3A. Proteins that were specifically enriched in PDE3A immunoprecipitates after treatment with DNMDP included Sirtuin 7 (SIRT7) and Schlafen 12 (SLFN12) (FIG. 13C and FIG. 14B). These proteins specifically interacted with PDE3A in the presence of DNMDP, and were not observed in the trequinsin treated control, whereas a known PDE3B interactor, abhydrolase domain-containing protein 15 (ABHD15, Chavez et al., Biochem. Biophys. Res. Commun. 342, 1218-1222, 2006), was enriched in the immunoprecipitate from trequinsin-treated cells (FIG. 13C and FIG. 14C). The interaction promoted by DNMDP between PDE3A and both SIRT7 and SLFN12 was validated with affinity reagents. Immunoprecipitation of endogenous PDE3A in HeLa cells treated with DNMDP, but not DMSO or trequinsin, enhanced complex formation of ectopically expressed V5-tagged SIRT7 and SLFN12 with PDE3A, as evidenced by coimmunoprecipitation (FIG. 19). DNMDP and (weakly) anagrelide, but not trequinsin, induced PDE3A and SFLN12 complex formation (FIG. 20). Without being bound to theory, PDE3A/SLFN12 complex formation correlated with cell killing (FIGS. 21A-21C).

Similar to PDE3A, overexpression of SLFN12 appears to have a cytotoxic effect in DNMDP sensitive cell lines, contributing to the difficulty of detecting SLFN12 in whole cell lysates.

The enhanced interaction of PDE3A with SIRT7 and SLFN12 indicated the possibility that one or more of these interacting proteins might contribute to DNMDP sensitivity. SIRT7 mRNA expression was relatively constant among all cells tested, but the co-expression of SLFN12 and PDE3A mRNA showed a strong correlation with DNMDP sensitivity; almost all DNMDP-sensitive cell lines expressed high levels of SLFN12 (FIG. 15A-15C). Importantly, almost half of sensitive cell lines expressing high levels of SLFN12 and PDE3A were found to be melanoma cell lines (FIG. 15B). SLFN12 expression alone was also one of the top genes correlating with sensitivity to DNMDP, corroborating the hypothesis that SLFN12 could be functionally involved in DNMDP-induced cytotoxicity (FIG. 16A). Moreover, when correcting for PDE3A expression, SLFN12 expression was the top correlating gene with DNMDP sensitivity (FIG. 16B). To assess whether SLFN12 is required for the cytotoxic phenotype of DMNDP, we reduced SLFN12 mRNA expression by 60% by knockdown with two shRNAs in HeLa cells (FIG. 15D). Similar to reduction in PDE3A expression, reduction of SLFN12 expression did not result in cytotoxicity, and in fact decreased sensitivity to DNMDP (FIG. 15E). These results show that SLFN12, like PDE3A, is required for the cytotoxic phenotype of DMNDP. Characterization of normal expression of SLFN12 and PDE3A by the GTEX consortium (Pierson, E. et al. PLoS Comput. Biol. 11, e1004220 (2015)) shows low expression of SLFN12 in normal tissues, while high co-expression of both PDE3A and SLFN12 is rarely observed (Table 3). This could suggest that on-target toxicity of DNMDP and related compounds may be potentially limited.

TABLE 3 RPKM values of SLFN12 and PDE3A expression in multiple healthy tissue types SLFN12 (RPKM) PDE3A (RPKM) Mean SD Mean SD n Adipose - Subcutaneous 2.14 0.70 4.76 2.03 128 Adipose - Visceral 2.43 1.03 4.26 1.94 31 (Omentum) Adrenal Gland 3.01 0.83 0.34 0.21 52 Artery - Aorta 2.10 0.71 16.15 5.12 82 Artery - Coronary 1.80 0.80 17.73 6.52 44 Artery - Tibial 1.09 0.49 22.97 6.35 137 Bladder 1.38 0.57 1.33 0.40 11 Brain - Amygdala 0.37 0.23 0.96 0.34 26 Brain - Anterior cingulate 0.28 0.16 0.77 0.45 22 cortex (BA24) Brain - Caudate (basal 0.40 0.23 1.27 0.37 36 ganglia) Brain - Cerebellar 0.11 0.07 2.73 1.49 29 Hemisphere Brain - Cerebellum 0.19 0.10 2.40 0.98 31 Brain - Cortex 0.25 0.12 0.56 0.59 25 Brain - Frontal Cortex 0.26 0.15 0.54 0.33 28 (BA9) Brain - Hippocampus 0.39 0.31 0.82 0.38 28 Brain - Hypothalamus 0.46 0.29 0.93 0.48 30 Brain - Nucleus accumbens 0.28 0.16 1.11 0.41 32 (basal ganglia) Brain - Putamen (basal 0.29 0.18 0.91 0.33 24 ganglia) Brain - Spinal cord 0.50 0.32 0.65 0.55 19 (cervical c-1) Brain - Substantia nigra 0.62 0.50 0.82 0.47 27 Breast - Mammary Tissue 2.48 0.74 3.19 2.35 66 Cells - EBV-transformed 4.70 1.57 0.02 0.01 54 lymphocytes Cells - Transformed 5.34 2.27 0.58 0.60 155 fibroblasts Colon - Sigmoid 1.58 0.50 10.27 3.45 13 Colon - Transverse 0.99 0.47 11.24 4.32 45 Esophagus - 1.14 0.31 16.87 5.53 22 Gastroesophageal Junction Esophagus - Mucosa 1.01 0.45 0.82 1.32 106 Esophagus - Muscularis 1.29 0.35 15.71 6.02 99 Fallopian Tube 2.32 0.86 3.80 1.86 6 Heart - Atrial Appendage 1.05 0.38 15.65 6.31 38 Heart - Left Ventricle 0.81 0.38 26.55 13.43 95 Kidney - Cortex 1.21 1.07 1.40 0.84 8 Liver 0.29 0.16 0.49 0.28 34 Lung 2.83 1.12 2.78 1.48 133 Minor Salivary Gland 1.75 0.61 0.62 0.44 5 Muscle - Skeletal 0.25 0.18 0.84 0.42 157 Nerve - Tibial 2.82 0.87 3.39 1.71 114 Ovary 1.92 0.57 2.17 1.13 35 Pancreas 0.52 0.27 2.65 0.86 65 Pituitary 0.47 0.23 1.04 0.47 22 Prostate 1.41 0.57 4.04 3.74 42 Skin - Not Sun Exposed 0.76 0.37 0.66 0.34 41 (Suprapubic) Skin - Sun Exposed (Lower 0.63 0.31 1.00 0.69 126 leg) Small Intestine - Terminal 1.61 0.72 7.34 4.83 17 Ileum Spleen 3.46 0.92 1.18 0.46 34 Stomach 1.10 0.40 3.93 5.35 81 Testis 0.49 0.19 0.43 0.20 60 Thyroid 3.19 0.96 2.59 1.34 120 Uterus 1.99 0.56 3.29 1.55 32 Vagina 1.39 1.39 2.49 2.49 34 Whole Blood 1.40 1.10 0.06 0.05 191

FIG. 22 shows that SLFN12 and CREB33L1 are lost in cells that have acquired resistance to DNMDP. Cell lines initially sensitive to DNMDP were made resistant by persistent exposure to DNMDP and subsequently analyzed by RNA-seq. Two genes were downregulated in both HeLa and H2122: SLFN 12 and CREB3L1. Accordingly, a reduction in levels of CREB33L1 and/or SLFN 12 indicates that cells have become resistant to DNMDP and other PDE3A modulators.

Two different cell lines, HeLa and H2122, made resistant to DNMDP by prolonged exposure, have commonly downregulated expression of two genes, SLFN12 and CREB3L1 (FIG. 22). Re-expression of SLFN12 restored sensitivity to DNMDP (FIG. 23A). Without being bound by theory, the restored sensitivity was dependent on PDE3A, as it was competed away by the PDE3A inhibitor, trequinsin. A DNMDP-resistant cell line A549 was sensitized by expression of SLFN12 or expression of SFLN12 and PDE3A (FIG. 23B). Expression of SLFN12 was sufficient to confer DNMDP sensitivity to A549 cells. Addition of PDE3A expression led to further sensitization.

Leiomyosarcomas are malignant smooth muscle tumors. Patient tumor samples from leiomyosarcomas were analyzed for PDE3A and SLFN12 expression to predict sensitivity of leiomyosarcomas (LMS) to DNMDP. Leiomyosarcomas are predicted to be sensitive to DNMDP due to prevalence among high purity TCGA samples expressing elevated levels of PDE3A and SLFN12 (FIG. 24, Table 4). P value for association of biomarker expression with leiomyosarcoma lineage: 0.0001.

TABLE 4 Leiomyosarcomas are predicted to be sensitive to DNMDP Predicted sensitive Predicted not sensitive LMS 17 31 Not LMS 38 1516

Differential scanning fluorimetry (DSF) was used to demonstrate binding of DNMDP to purified PDE3A catalytic domain, PDE3A(677-1141). In this experiment, 5 μM hsPDE3A(640-1141) was incubated in the absence or presence of 100 μM compounds, as indicated in Table 5. Binding buffer: 20 mM Hepes pH 7.4, 100 μM TCEP, 1 mM MgCl₂, 150 mM NaCl.

TABLE 5 Binding of DNMDP to PDE3A(677-1141) T_(m) (° C.) ΔT_(m) (° C.) PDE3A₆₇₇₋₁₁₄₁ 52.4 ± 0.0 PDE3A₆₇₇₋₁₁₄₁ + DNMDP 58.4 ± 0.0 6.0 PDE3A₆₇₇₋₁₁₄₁ + Anagrelide 56.6 ± 0.0 4.2 PDE3A₆₇₇₋₁₁₄₁ + Trequinsin 66.2 ± 0.0 14.2 PDE3A₆₇₇₋₁₁₄₁ + (Compound 3) 59.0 ± 0.0 6.6

Using predictive chemogenomics, a class of compounds was discovered, exemplified by DNMDP, that targeted a novel cancer dependency by small-molecule modulation of PDE3A. These compounds bound PDE3A in a mutually exclusive manner with non-cytotoxic PDE3 inhibitors and exerted a neomorphic or hypermorphic effect on the function of PDE3A, leading to a change in its protein-protein interactions. One unique protein-interaction partner, SLFN12, was highly expressed in DNMDP-sensitive cell lines, indicating a functional role in the pathway through which the cytotoxic signal was relayed. As a result, DNMDP was both selective and potent across a large panel of cancer cell lines.

Here, a novel cytotoxic compound was identified with great selectivity and low-nM potency against cancer cell lines across multiple lineages. Using gene-expression correlates for predictive chemogenomics, PDE3A was identified as the putative target of this small molecule, DNMDP. Interestingly, loss of PDE3A expression resulted in resistance to DNMDP. Moreover, PDE3A immunoprecipitation followed by isobaric stable isotope tags for relative abundance and quantitation by mass spectrometry (iTRAQ/MS) identified SLFN12 and SIRT7 as novel protein-protein interaction partners of PDE3A upon DNMDP binding, possibly due to allosteric modulation of the function of PDE3A. Importantly, SLFN12 expression was the top correlating gene with DNMDP sensitivity when corrected for PDE3A expression. Single gene or multi-gene expression correlations have shown to help elucidate the mechanism of action and relevant signaling pathways of small molecules. A novel biochemical target for cancer treatment was identified that is unlikely to have been found by target identification approaches such as loss-of-function screens or genomic analysis.

PDE3A belongs to the superfamily of phosphodiesterases and together with PDE3B forms the PDE3 family. The PDE3 family has dual substrate affinity and hydrolyses both cAMP and cGMP. Expression of PDE3A is highest in the cardiovascular system, platelets, kidney, and oocytes (Ahmad et al., Horm Metab Res 44, 776-785, 2012). The clinical PDE3 inhibitor cilostazol has been developed to treat intermittent claudication, as PDE3A inhibition in platelets impairs activation and platelet coagulation (Bedenis et al., Cochrane Database Syst Rev 10, CD003748, 2014). Other PDE3 inhibitors, such as milrinone, amrinone, and levosimendan, are indicated to treat congestive heart failure, where the combination of vasodilation and elevated cardiac cAMP levels increases cardiac contractility (Movsesian et al., Curr Opin Pharmacol 11, 707-713, 2011). None of these clinical inhibitors were able to replicate the cytotoxic phenotype of DNMDP, indicating that cyclic nucleotide hydrolysis was not sufficient to induce cell death in DNMDP-sensitive cell lines.

Interestingly however, other PDE3 inhibitors such as zardaverine, anagrelide, and quazinone have been reported previously to have cell cytotoxic characteristics in a select number of cancer cell lines (Sun et al., PLoS ONE 9, e90627, 2014; Fryknas et al., J Biomol Screen 11, 457-468, 2006). In concordance with the present findings, other PDE3 and PDE4 inhibitors were found not to replicate the cytotoxic phenotype of zardaverine where retinoblastoma protein retinoblastoma 1 (RB1) expression was reported to separate zardaverine sensitive cell lines from non-sensitive cell lines (Sun et al., PLoS ONE 9, e90627, 2014). This finding was in contrast to the present data where a correlation between cytotoxic activities of DNMDP and copy-number or mRNA expression of RB was not identified. Another PDE3 inhibitor, anagrelide, uniquely inhibited megakaryocyte differentiation, resulting in apoptosis. Other PDE3 inhibitors tested did not have this activity (Wang et al., Br. J. Pharmacol. 146, 324-332, 2005; Espasandin, Y. et al., J. Thromb. Haemost. n/a-n/a, 2015, doi:10.1111/jth.12850). It was hypothesized that the reported effects of zardaverine on cell viability and anagrelide on megakaryocyte differentiation are mediated through the same PDE3A modulation as described in this study.

Multiple PDE3 inhibitors were competitive inhibitors and have been shown to occupy the catalytic binding site of cAMP and cGMP (Card et al., Structure 12, 2233-2247, 2004; Zhan et al., Mol. Pharmacol. 62, 514-520, 2002). In addition, zardaverine has been co-crystalized in a complex with PDE4D, where it occupies the cAMP-binding site, and has been modeled to bind PDE3B in a similar manner (Lee et al., FEBS Lett. 530, 53-58, 2002). Given the structural similarity of DNMDP to zardaverine and that DNMDP inhibited both PDE3A and PDE3B, it was hypothesized that the binding mode of DNMDP is very similar to that of zardaverine. This indicated that in addition to acting as a cAMP/cGMP-competitive inhibitor, DNMDP allosterically induces a conformation that is responsible for its cytotoxic phenotype. Allosteric modulation of phosphodiesterases has been described previously for PDE4, where small molecules bound in the active site and simultaneously interacted with regulatory domains that came across the PDE4 active site. As a result, allosteric modulators stabilized a protein conformation that has been shown to differentially bind different PDE4 partner proteins (Burgin et al., Nat Biotechnol 28, 63-70, 2010).

The study of proteins associated with PDE3A might illuminate both its normal function and the way in which PDE3A modulators such as DNMDP kill cancer cells. PDE3A interacted with protein phosphatase 2 subunits, which are implicated in oncogenic viral transformation and are mutated in human cancers (Nagao et al., Int. Symp. Princess Takamatsu Cancer Res. Fund 20, 177-184, 1989; Imielinski et al., Cell 150, 1107-1120, 2012; Lawrence et al., Nature 499, 214-218, 2013), indicating a role for PDE3A in cancer cell signaling. Even though these interactions were not induced by DNMDP binding, the importance of the protein phosphatases in cancer biology would warrant further research.

The enhanced interaction between PDE3A and SLFN12, facilitated by DNMDP binding to PDE3A, and the correlation between sensitivity to DNMDP with SLFN12 expression strongly indicated that it is necessary to understand the functional impact of the PDE3A-SLFN12 interaction. However, little is known at this time about the functional role of SLFN12 in human physiology and cancer biology. SLFN12 is part of the schlafen gene family that diverges largely between humans and rodents. The large difference is due to rapid gene evolution and positive selection (Bustos et al., Gene 447, 1-11, 2009). Therefore, SLFN12 has no murine orthologue, preventing the study of SLFN12 in a well-understood model organism. The single publication on SLFN12 showed modulation of prostate cancer cell lines after ectopic expression of SLFN12 (Kovalenko et al., J. Surg. Res. 190, 177-184, 2014). Additional studies into the function of SLFN12 and its interaction with PDE3A could elucidate the mechanism of DNMDP cytotoxicity. Two observations indicated that DNMDP acted as a neomorph or hypermorph on PDE3A function: 1) DNMDP-sensitive cancer cell lines did not depend on PDE3A expression for survival, but rather PDE3A knock-down led to DNMDP resistance; and 2) DNMDP induced or enhanced protein-protein interactions upon binding to PDE3A. Lenalidomide was an example of a small molecule that acted as a neomorph or hypermorph rather than as an enzymatic inhibitor. Lenalidomide modulated a specific protein-protein interaction between the cereblon ubiquitin ligase and Ikaros transcription factors, which were then subsequently targeted for degradation (Krönke et al., Science 343, 301-305, 2014; Lu et al., Science 343, 305-309, 2014). By analogy, DNMDP might directly stabilize a PDE3A-SLFN12 interaction, or DNMDP could allosterically stabilize a PDE3 conformation that binds SLFN12. Either of these mechanisms could result in a neo- or hypermorphic phenotype. Further characterization of the neomorphic phenotype induced by DNMDP might facilitate synthesis of small molecules that will not inhibit cyclic nucleotide hydrolysis by PDE3A. Toxicity profiles of such small molecules should differ from PDE3 inhibitors prescribed for cardiovascular indications.

This study has uncovered a previously unknown role for PDE3A in cancer maintenance, in which its function can be modified by a subset of PDE3 inhibitors, resulting in toxicity to a subset of cancer cell lines. These data indicated that DNMDP and its analogs had a hyper- or neomorphic effect on PDE3A, leading to cellular toxicity, which was corroborated by cells becoming less sensitive to DNMDP with decreasing levels of cellular PDE3A. These observations are comparable with other reports of allosteric modulation of phosphodiesterases (Burgin et al., Nat Biotechnol 28, 63-70, 2010), indicating that DNMDP and analogues may have similar effects on PDE3A. The exact mechanism of cell-selective cytotoxicity remains unknown for now; however, further studies into the novel interactions with SLFN12, and perhaps SIRT7, might be informative.

In summary, the study herein used differential cytotoxicity screening to discover a cancer cell cytotoxic small molecule, DNMDP. Profiling of DNMDP in 766 genomically-characterized cancer cell lines revealed stereospecific nanomolar efficacy in about 3% of cell lines tested. A search for genomic features that predicted sensitivity revealed that elevated PDE3A expression strongly correlated with DNMDP response. DNMDP inhibited PDE3A and PDE3B, with little or no activity towards other PDEs. However, unexpectedly, most other PDE3A inhibitors tested did not phenocopy DNMDP, including the potent and selective PDE3A inhibitor, trequinsin. Co-treatment of DNMDP-sensitive cells with trequinsin competed away the cancer cell cytotoxic activity of DNMDP, and knockout of PDE3A rescued the otherwise sensitive cells from DNMDP-induced cytotoxicity, leading us to hypothesize that PDE3A is required for cancer cell killing by DNMDP, which induces a neomorphic alteration of PDE3A. Mass spectrometric analysis of PDE3A immunoprecipitates alone or in the presence of DNMDP or trequinsin revealed differential binding of SLFN12 and SIRT7 only in the presence of DNMDP. Similar to PDE3A, SLFN12 expression levels were elevated in DNMDP-sensitive cell lines, and knock down of SLFN12 with shRNA decreased sensitivity of cells to DNMDP, indicating that DNMDP-induced complex formation of PDE3A with SLFN12 is critical to the cancer cell cytotoxic phenotype. Results herein therefore implicate PDE3A modulators as candidate cancer therapeutic agents and demonstrate the power of predictive chemogenomics in small molecule discovery.

The Experiments Above were Performed with the Following Methods and Materials:

Compound Library Screening in NCI-H1734 and A549 Cell Lines

1500 NCI-H1734 or 1000 A549 cells were plated in a 384-well plate in 40 μl of RPMI supplemented with 10% Fetal Bovine Serum and 1% Pen/Strep. 24 hours after plating, a compound-library of 1924 small molecules was added at a concentration of 1 μM. Staurosporine was used a positive control for cytotoxicity at a concentration of 1 μM, and DMSO was used a negative control at a concentration of 1%. All compounds were incubated for 48 hours with indicated small molecules. After 48 hours, 384-well plates were removed from the incubator and allowed to cool to room temperature for 20 minutes. Cell viability was assessed by adding 40 μl of a 25% CELLTITERGLO® (Promega) in PBS with a THERMO COMBI™ or multichannel-pipette and incubated for 10 minutes. The luminescence signal was read using a Perkin-Elmer EnVision. Viability percentage was calculated by normalizing to DMSO controls.

Compound Sensitivity Testing in Cell Lines

1000 HeLa (DMEM), 1000 A549 (RPMI), 500 MCF-7 (DMEM), 4000 PC3 (F12-K), 1000 NCI-H2122 (RPMI) or 1500 NCI-H1563 (RPMI) cells were plated in a 384-well plate in 40 g of corresponding growth media supplemented with 10% Fetal Bovine Serum. 24 hours after plating, indicated compounds were added at indicated concentrations and incubated for 48 hours. Cell viability was assessed as described in Compound library screening in NCI-H1734 and A549 cell lines. Compound 6 was tested in the HeLa cell viability assay and its EC50 was determined to be 1.1 nM.

Caspase Activity in HeLa Cells

1000 HeLa cells were plated in 384-well plate in 40 g of corresponding growth media supplemented with 10% Fetal Bovine Serum. 24 hours after plating, indicated compounds were added at indicated concentrations and incubated for 48 hours. Caspase-Glo from Promega was added according to the manufacturers recommendations and luminescence was determined as described in Compound library screening in NCI-H1734 and A549 cell lines.

Large-Scale Cell-Line Viability Measurements

The sensitivity of 777 cancer cell lines (CCLs) was measured drawn from 23 different lineages to DNMDP. Cancer cell lines are part of the Cancer Cell Line Encyclopedia and have their identities confirmed through SNP arrays and somatic DNA alterations. Each cell line was plated in its preferred media in white opaque 1536-plates at a density of 500 cells/well. After incubating overnight, DNMDP was added by acoustic transfer at 16 concentrations ranging from 66.4 μM-2 nM in 2-fold steps in duplicate (Labcyte Echo 555, Labcyte Inc., Sunnyvale, Calif.). After 72 hours treatment, cellular ATP levels were measured as a surrogate for viability (CELLTITERGLO®, Promega Corporation, Madison, Wis.) according to manufacturer's protocols using a ViewLux Microplate Imager (PerkinElmer, Waltham, Mass.) and normalized to background (media-only) and vehicle (DMSO)-treated control wells.

Concentration response curves were fit using nonlinear fits to 2- or 3-parameter sigmoid functions through all 16 concentrations with the low-concentration asymptote set to the DMSO-normalized value, and an optimal 8-point dose curve spanning the range of compound-sensitivity was identified. The area under the 8-point dose curve (AUC) was computed by numeric integration as a metric for sensitivity for further analysis. Similar sensitivity measurements have been obtained for a collection of 480 other compounds, enabling analyses that identify cell lines responding uniquely to DNMDP (see Broad Institute Cancer Therapeutics Response Portal, a dataset to identify comprehensively relationships between genetic and lineage features of human cancer cell lines and small-molecule sensitivities, for the complete list of compounds).

Correlation of Sensitivity Measurements with Basal Gene Expression

Gene-centric robust multichip average (RMA)-normalized basal mRNA gene expression data measured on the Affymetrix GeneChip Human Genome U133 Plus 2.0 Array were downloaded from the Cancer Cell Line Encyclopedia (CCLE, a detailed genetic characterization of a large panel of human cancer cell lines; Barretina et al., Nature 483, 603-607, 2012). Pearson correlation coefficients were calculated between gene expression (18,988 transcripts) and areas under the curve (AUCs) across 760 overlapping CCLs. For comparisons across small molecules exposed to differing numbers of CCLs, correlation coefficients were transformed using Fisher's transformation.

Method for PDE3A Enzyme Inhibition

The commercially available 3H-cAMP Scintillation Proximity Assay (SPA, Perkin Elmer) system was used for enzyme inhibition studies. For the determination of the in vitro effect of test substances on the PDE3A reactions 2 μl of the respective test compound solution in DMSO (serial dilutions) were placed in wells of microtiter plates (Isoplate-96/200W; Perkin Elmer). 50 μl of a dilution of PDE3A cell extract from Sf9 cells overexpressing human full length PDE3A (SB Drug Discovery, UK) in buffer A (50 mM Tris/HCl pH 7.5, 8.3 mM MgCl₂, 1.7 mM EDTA, 0.2% BSA) was added. The dilution of the PDE3A cell extract was chosen such that the reaction kinetics was linear and less than 70% of the substrate was consumed (typical dilution 1:5000). The reaction was started by addition of 50 μl (0.025 μCi) of 1:2000 in buffer A w/o BSA diluted substrate [8-³H] adenosine 3′, 5′-cyclic phosphate (1 μCi/μl; Perkin Elmer). After incubation at room temperature for 60 min, the reaction was stopped by addition of 25 μl of a suspension containing 18 mg/ml yttrium scintillation proximity beads (Perkin Elmer) in water. The microtiter plates were sealed and measured in a Microbeta scintillation counter (PerkinElmer Wallac). IC₅₀ values were determined from sigmoidal curves by plotting percentage PDE3A activity vs log compound concentration. For compound 6 the IC50 values are 2.4 nM (PDE3A IC50) and 3.4 nM (PDE3B IC50) respectively.

Method for Human Cryo Hepatocytes:

Investigation of In Vitro Metabolic Stability in Cryopreserved Human Hepatocytes (Including Calculation of Hepatic In Vivo Blood Clearance (CL) and Maximal Oral Bioavailability (Fmax))

Cryopreserved Hepatocytes (e.g. purchased from Celsis InVitroTechnologies) were briefly thawed, washed with 45 mL pre-warmed in in vitro GRO HT medium and centrifuged for 5 min at 50×g. The cell pellet was resuspended in 5 ml of Krebs-Henseleit Butter (KHB). Cell viability was determined by trypan blue exclusion.

For the metabolic stability assay liver cells were distributed in WME containing 5% FCS to glass vials at a density of 1.0×106 vital cells/ml. The test compound was added to a final concentration of 1 μM. During incubation, the hepatocyte suspensions were continuously shaken at 580 rpm and aliquots were taken at 2, 8, 16, 30, 45 and 90 min, to which equal volumes of cold methanol were immediately added. Samples were frozen at −20° C. over night, after subsequently centrifuged for 15 minutes at 3000 rpm and the supernatant was analyzed with an Agilent 1290 HPLC-system with LCMS/MS detection.

The half-life of a test compound was determined from the concentration-time plot. From the half-life the intrinsic clearances were calculated. Together with the additional parameters liver blood flow, amount of liver cells in vivo and in vitro. The hepatic in vivo blood clearance (CL) and the maximal oral bioavailability (Fmax) was calculated. The hepatic in vivo blood clearance (CLblood) and the maximal oral bioavailability (Fmax) was calculated using the following formulae: CL′intrinsic [ml/(min*kg)]=kel [1/min]/((cellno/volume of incubation [ml])*fu,inc)*(cellno/liver weight [g])*(specific liver weight [g liver/kg body weight]); CLblood well-stirred [L/(h*kg)]=(QH [L/(h*kg)]*fu,blood*CL′intrinsic [L/(h*kg)])/(QH [L/(h*kg)]+fu,blood*CL′intrinsic [L/(h*kg)]); Fmax=1−CLblood/QH and using the following parameter values: Liver blood flow−1.32 L/h/kg human; specific liver weight−21 g/kg body weight; liver cells in vivo—1.1×108 cells/g liver, liver cells in vitro−1.0×106/ml.; fu,inc and fu,blood is taken as 1.

(5R)-6-[3-chloro-5-fluoro-4-(morpholin-4-yl)phenyl]-5-methyl-4,5-dihydropyridazin-3(2H)-one (Compound 6,) displays increased stability in human Hepatocytes (mean metabolic stability (Fmax)=93%) in comparison to (5R)-6-[3-fluoro-4-(morpholin-4-yl)phenyl]-5-methyl-4,5-dihydropyridazin-3(2H)-one (Compound 3 of WO 2014/164704) (mean metabolic stability (Fmax)=49%).

Chemistry Experimental Methods General Details

All reactions were carried out under nitrogen (N2) atmosphere. All reagents and solvents were purchased from commercial vendors and used as received. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker (300 or 400 MHz ¹H, 75 or 101 MHz ¹³C) spectrometer. Proton and carbon chemical shifts are reported in ppm (δ) referenced to the NMR solvent. Data are reported as follows: chemical shifts, multiplicity (br=broad, s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet; coupling constant(s) in Hz). Flash chromatography was performed using 40-60 μm Silica Gel (60 Å mesh) on a Teledyne Isco Combiflash Rf. Tandem Liquid Chromatography/Mass Spectrometry (LC/MS) was performed on a Waters 2795 separations module and 3100 mass detector with a Waters Symmetry C18 column (3.5 μm, 4.6×100 mm) with a gradient of 0-100% CH3CN in water over 2.5 min with constant 0.1% formic acid. Analytical thin layer chromatography (TLC) was performed on EM Reagent 0.25 mm silica gel 60-F plates. Elemental analysis was performed by Robertson Microlit Laboratories, Ledgewood N.J.

Synthesis of (R)-DNMDP

In 5 mL of acetic anhydride, 2.00 g (9.84 mmol) of (R)-6-(4-aminophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one (A, Toronto Research Chemicals) was stirred 1 hour before addition of 30 mL water, filtration, rinsing the solids with water and drying to yield 2.20 g of product B (91%). ¹H NMR (300 MHz, DMSO-d₆) δ 10.92 (s, 1H), 10.13 (s, 1H), 7.74 (d, J=8.9, 2H), 7.65 (d, J=8.8, 2H), 3.41-3.33 (m, 1H), 2.68 (dd, J=6.8, 16.8, 1H), 2.23 (d, J=16.7, 1H), 2.08 (s, 3H), 1.07 (d, J=7.3, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ 168.50, 166.27, 152.25, 140.27, 129.24, 126.24, 118.70, 33.47, 26.91, 24.02, 15.87. HPLC: R_(t) 0.72 min, purity >95%. MS: 246 (M+1).

To 3.09 g of B (15.3 mmol) dissolved in 30 mL of sulfuric acid and cooled in an ice bath was added 0.72 mL of 90% nitric acid (15 mmol) in 8 mL sulfuric acid via an addition funnel over 10 minutes. After stirring 1 hour the mixture was poured onto ice. The yellow solid was filtered off and the water was rinsed several times with EtOAc before drying and combining with the yellow solid. Chromatography with 40-60% EtOAc in hexane yielded 1.12 g (25%) of product as a yellow solid which was recrystallized from EtOAc. ¹H NMR (300 MHz, DMSO-d₆) δ 11.13 (s, 1H), 10.41 (s, 1H), 8.25 (d, J=1.8, 1H), 8.07 (dd, J=1.8, 8.6, 1H), 7.71 (d, J=8.6, 1H), 3.55-3.40 (m, 1H), 2.74 (dd, J=6.9, 16.8, 1H), 2.27 (d, J=16.8, 1H), 2.09 (s, 3H), 1.08 (d, J=7.2, 3H). ¹³C NMR (75 MHz, DMSO-d6) δ 168.57, 166.31, 150.37, 142.19, 131.69, 131.32, 130.60, 125.07, 121.70, 33.30, 26.81, 23.44, 15.64. TLC: Rf 0.25 (1:1 EtOAc:hexane). HPLC: R_(t) 0.87 min, purity >95%. MS: 291 (M+1). HRMS Exact Mass (M+1): 291.1088. Found: 291.1091

To 58 mg of C (0.20 mmol) dissolved in 10 mL of MeOH was added a solution of 48 mg NaOH (1.2 mmol) in 0.5 mL water. After 1 hour the reaction was concentrated, water was added and rinsed with EtOAc, the EtOAc was dried and concentrated to give 48 mg (93%) of product D. ¹H NMR (300 MHz, DMSO-d6) δ 10.92 (s, 1H), 8.28 (d, J=2.0, 1H), 7.87 (dd, J=2.1, 9.0, 1H), 7.76 (s, 2H), 7.06 (d, J=9.0, 1H), 3.33 (s, 1H), 2.67 (dd, J=6.8, 16.8, 1H), 2.22 (d, J=16.6, 1H), 1.06 (d, J=7.3, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ 166.25, 151.12, 146.69, 132.72, 129.80, 122.57, 122.19, 119.80, 33.43, 26.70, 15.77. MS: 249 (M+1).

To 35 mg of amine D (0.14 mmol) dissolved in 0.5 mL Dimethylformamide (DMF) was added 70 mg of acetaldehyde (1.6 mmol) and 170 mg of NaBH(OAc)₃ (0.80 mmol) and 10 μL, (0.2 mmol) of HOAc. After stirring 3 hours, water and EtOAc were added, the EtOAc separated, dried, concentrated and chromatographed with 30-50% EtOAc in hexane to isolate 3 mg of the (R)-DNMDP (7%). The synthesized material was identical to purchased racemic material by TLC, HPLC and ¹H NMR. ¹H NMR (300 MHz, CDCl₃) δ 8.58 (s, 1H), 8.04 (d, J=2.3, 1H), 7.84 (dd, J=2.3, 9.0, 1H), 7.11 (d, J=9.0, 1H), 3.30-3.36 (m, 1H), 3.26 (q, J=7.1, 4H), 2.71 (dd, J=6.8, 16.9, 1H), 2.48 (d, J=17.0, 1H), 1.25 (d, J=7.4, 3H), 1.16 (t, J=7.1, 6H). TLC: Rf 0.25 (1:1 EtOAc:hexane). HPLC: R_(t) 1.27 min, purity >95%. MS: 305 (M+1). Exact Mass (M+1): 305.1608 Found: 305.1616. ¹³C NMR (75 MHz, CDCl₃, purchased material) δ 166.28, 152.02, 145.24, 141.21, 129.77, 124.94, 123.94, 121.00, 46.10, 33.80, 27.81, 16.24, 12.56.

The optical purity of (R)-DNMDP was determined using chiral SCF chromatography and comparison to commercially available racemic material: Column: ChiralPak AS-H, 250×4.6 mm, 5 μm, Mobile Phase Modifier: 100% Methanol, Gradient: 5 to 50% Methanol over 10 minutes, Flow Rate: 4 mL/min, Back Pressure: 100 bar, Column Temperature: 40 C. UV detection was from 200-400 nm. Retention times of separated isomers: 5.36, 6.64 minutes; retention time of (R)-DNMDP, 6.60 minutes, 1:19 ratio of enantiomers detected.

2. To 200 mg (0.98 mmol) of A dissolved in 5 mL of MeOH was added 87 mg of acetaldehyde (2.0 mmol), 113 uL of HOAc (2.0 mmol) and 124 mg (2.0 mmol) of NaBH₃CN and the reaction was stirred overnight at room temperature. The next day the same quantity of reagents were added and the reaction stirred another 24 hours. The mixture was concentrated and partitioned between CH₂Cl₂ and water, the CH₂Cl₂ was separated, dried, and concentrated before chromatography with 20-40% EtOAc in hexane isolated 210 mg of product as a white solid (82%). ¹H NMR (300 MHz, CDCl₃) δ 8.95 (s, 1H), 7.64 (d, J=8.7, 2H), 6.66 (d, J=8.7, 2H), 3.37 (dd, J=9.6, 16.4, 5H), 2.67 (dd, J=6.5, 16.8, 1H), 2.43 (d, J=16.8, 1H), 1.41-1.02 (m, 10H). ¹³C NMR (75 MHz, CDCl₃) δ 166.82, 154.55, 148.79, 127.32, 120.81, 111.08, 44.32, 33.92, 27.74, 16.37, 12.50. TLC: Rf 0.25 (1:1 EtOAc:hexane). HPLC: R_(t) 1.05 min, purity >95%. MS: 260 (M+1). HRMS Exact Mass (M+1): 260.1757. Found: 260.1764

3. To 200 mg (0.984 mmol) of A dissolved in 1 mL of Dimethylformamide (DMF) was added 250 μL (2.00 mmol) of bis (2-bromoethyl) ether and 400 mg of K2CO3 and the mixture was stirred overnight at 60° C. The next day another 250 μL of bis (2-bromoethyl) ether and 170 mg of K2CO3 were added. After 3 hours, EtOAc and water were added, the water was rinsed with EtOAc, the combined EtOAc washes were dried and concentrated. Chromatography with 0-4% MeOH in CH2Cl2 yielded 125 mg of product (46%). ¹H NMR (300 MHz, CDCl₃) δ 8.61 (s, 1H), 7.68 (d, J=8.8, 2H), 6.92 (d, J=8.8, 2H), 3.99-3.76 (m, 4H), 3.44-3.31 (m, 1H), 3.29-3.22 (m, 4H), 2.70 (dd, J=6.7, 16.8, 1H), 2.46 (d, J=16.7, 1H), 1.24 (d, J=7.3, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 166.64, 154.05, 152.18, 127.10, 125.33, 114.73, 66.69, 48.33, 33.93, 27.94, 16.36. TLC: R_(f) 0.1 (1:50 MeOH:CH₂Cl₂). HPLC: R_(t) 1.05 min, purity >95%. MS: 274 (M+1). HRMS: calcd. 274.1556 (M+1); found 274.1552. Anal. Calcd. for C₁₅H₁₉N₃O₂: C, 65.91; H, 7.01; N, 15.37; Found. 65.81, H, 6.66, N, 15.26.

DNMDP-2L. To 130 mg of A (0.64 mmol) dissolved in 0.4 mL of Dimethylformamide (DMF) was added 100 mg of tert-butyl 2-(2-(2-bromoethoxy)ethoxy)-ethylcarbamate (Toronto Research Chemical, 0.32 mmol) and 90 mg of K₂CO³ (64 mmol) and the mixture was stirred at 60° C. overnight. After cooling, water was added and rinsed several times with EtOAc. The combined EtOAc layers were dried, concentrated, and chromatographed with 50-70% EtOAc to yield 81 mg of product (58%). ¹H NMR (300 MHz, CDCl₃) δ 9.06 (s, 1H), 7.59 (d, J=8.8 Hz, 2H), 6.62 (d, J=8.8 Hz, 2H), 5.15 (s, 1H), 4.53 (s, 1H), 3.72 (t, J=5.2 Hz, 2H), 3.65 (s, 4H), 3.55 (t, J=5.2 Hz, 2H), 3.32 (m, 5H), 2.67 (dd, J=16.8, 6.7 Hz, 1H), 2.42 (d, J=16.4 Hz, 1H), 1.44 (s, 9H), 1.22 (d, J=7.4 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 166.83, 155.99, 154.45, 149.64, 127.33, 123.24, 112.58, 79.28, 70.30, 70.26, 70.22, 69.45, 43.14, 40.39, 33.96, 28.43, 27.89, 16.40; HPLC: R_(t) 2.50 min (7.5 min run), purity >95%. MS: 435 (M+1). This product (0.19 mmol) was dissolved in 1 mL MeOH and to the solution was added acetaldehyde (50 uL, 0.89 mmol), 10 uL HOAc (0.2 mmol) and 12 mg NaBH₃CN (0.19 mmol). After 1 hour, NaHCO₃(aq) and CH₂Cl₂ were added, the CH₂C2 was separated and the water washed twice with CH₂C2. The combined CH₂Cl₂ was dried, concentrated, and chromatography with 60-70% EtOAc in hexane yielded 71 mg of product as a clear oil (82%). ¹H NMR (400 MHz, CDCl₃) δ 8.91 (s, 1H), 7.63 (d, J=8.9 Hz, 2H), 6.69 (d, J=8.9 Hz, 2H), 5.07 (s, 1H), 3.65 (t, J=6.0 Hz, 2H), 3.61 (s, 4H), 3.55 (dt, J=9.9, 5.5 Hz, 4H), 3.46 (q, J=7.0 Hz, 2H), 3.38-3.22 (m, 3H), 2.67 (dd, J=16.8, 6.7 Hz, 1H), 2.43 (d, J=16.7 Hz, 1H), 1.45 (s, 10H), 1.23 (d, J=7.3 Hz, 3H), 1.18 (t, J=7.0 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 166.84, 155.96, 154.46, 148.89, 127.35, 121.38, 111.28, 79.22, 70.68, 70.27, 70.24, 68.74, 49.95, 45.49, 40.32, 33.97, 28.43, 27.80, 16.43, 12.14. R_(t) 2.99 min (7.5 min run), purity >95%. MS: 463 (M+1).

Synthesis of Compound 6 A. Compound 3

Compound 3 could be obtained via two different routes:

or

1-(3-Fluoro-4-morpholinophenyl)propan-1-one

To a 1 L one-neck flask was added 40 g of 3,4-difluoropropiophenone (235 mmol), 400 mL of CH₃CN, 250 mL of morpholine (2.86 mol), and 50 mL of DIPEA (360 mmol) and the solution was heated at 100° C. overnight. The next day the reaction was cooled and concentrated. The mixture was dissolved in CH₂Cl₂ and rinsed several times with water, then brine, and was dried (MgSO₄), filtered and concentrated. Most of the crude product dissolved in approx. 1 L of hot hexane and was cooled overnight. Upon filtration, more crystals appeared in the mother liquors. The mother liquors were concentrated and recrystallized from hexane. A total of 52.5 g of dry white solid was obtained (94%).

¹H NMR (400 MHz, CDCl₃) δ 7.72 (dd, J=8.4, 1.9 Hz, 1H), 7.66 (dd, J=14.0, 2.0 Hz, 1H), 6.93 (t, J=8.5 Hz, 1H), 3.94-3.85 (m, 4H), 3.26-3.17 (m, 4H), 2.94 (q, J=7.3 Hz, 2H), 1.23 (t, J=7.3 Hz, 3H). ¹⁹F NMR (376 MHz, CDCl₃) δ −121.48. MS: 238 (M+1).

Ethyl 4-(3-fluoro-4-morpholinophenyl)-3-methyl-4-oxobutanoate

To a 2 L three-necked flask was added 200 mL of anhydrous THF and 200 mL of LiHMDS solution (1 M in THF) and the flask was cooled on a dry ice/isopropanol bath. Once cold, 46.5 g of 3-fluoro-4-morpholino)propiophenone (196 mmol) dissolved in 300 mL of THF was added via cannula. After stirring 1 h, 44 mL of ethyl bromoacetate (202 mmol) dissolved in 44 mL of THF was added, and the reaction mixture was stirred overnight, warming to room temperature. The next morning, the reaction was still a little cold. To it was added NH₄Cl solution, followed by EtOAc and hexane. The layers were separated, the aqueous layer was rinsed twice with EtOAc, the combined organic layers were rinsed with brine, dried (MgSO₄), and concentrated to 65.6 g of pale yellow oil (100%) which was carried on crude.

¹H NMR (400 MHz, CDCl₃) δ 7.74 (dd, J=8.4, 1.8 Hz, 1H), 7.67 (dd, J=14.1, 1.9 Hz, 1H), 6.93 (t, J=8.5 Hz, 1H), 4.09 (q, J=6.9 Hz, 2H), 3.93-3.78 (m, 5H), 3.28-3.14 (m, 4H), 2.93 (dd, J=16.8, 8.5 Hz, 1H), 2.43 (dd, J=16.8, 5.6 Hz, 1H), 1.21 (dt, J=7.1, 3.6 Hz, 6H). ¹⁹F NMR (376 MHz, CDCl₃) δ −121.33. ¹H NMR, ¹⁹F NMR, and LC indicated impurities were 5-10%.

5R)-6-[3-fluoro-4-(morpholin-4-yl)phenyl]-5-methyl-4,5-dihydropyridazin-3(2H)-one Compound 3a (Compound 3-racemate)

The crude ethyl 4-(3-fluoro-4-morpholinophenyl)-3-methyl-4-oxobutanoate (65.6 g, 202 mmol) was dissolved in 400 mL of EtOH and to it was added 31.6 mL of hydrazine (1.01 mol) and the reaction was heated at reflux temperature overnight. The next morning, much white precipitate was present in the flask which was cooled to room temperature, the solids were filtered and rinsed with cold EtOH. The solids were put in a 1 L one-neck flask and placed on a rotary evaporator to remove residual EtOH, 42 g of clean solid was obtained (71%).

¹H NMR (300 MHz, CDCl₃) δ 8.46 (s, 1H), 7.50 (dd, J=2.1, 14.4, 1H), 7.43 (dd, J=1.8, 8.4, 1H), 6.93 (t, J=8.7, 1H), 3.95-3.83 (m, 4H), 3.37-3.23 (m, 1H), 3.21-3.11 (m, 4H), 2.70 (dd, J=6.8, 16.9, 1H), 2.47 (d, J=17.1, 1H), 1.24 (d, J=7.4, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 166.45, 155.33 (d, J_(C-F)=246.3), 152.71, 141.1 (d, J_(C-F)=8.6), 128.75 (d, J_(C-F)=7.6), 122.20 (d, J_(C-F)=3.0), 118.09 (d, J_(C-F)=3.6), 113.80 (d, J_(C-F)=23.0), 66.83, 50.50 (d, J_(C-F)=3.9), 33.84, 27.96, 16.29. ¹⁹F NMR (282 MHz, CDCl₃) δ −121.51. Mass: 292 (M+1).

Analytical Separation Method

Instrument: Thar analytical SFC

Column: ChiralPak AS-H, 250×4.6 mm

Mobile phase: A for C02 and B for MeOH (0.05% DEA)

Gradient: B 40%

Flow rate: 2.4 mL/min

Back pressure: 100 bar

Column temperature: 35° C.

Wavelength: 220 nm

Preparative Separation Method

Instrument: Thar200 preparative SFC

Column: ChiralPak AS-10 m, 300×50 mmI.D.

Mobile phase: A for CO2 and B for EtOH (0.1% NH3.H2O)

Gradient: B 45%

Flow rate: 200 mL/min

Back pressure: 100 bar

Column temperature: 38° C.

Wavelength: 220 nm

Sample Preparation:

Compound 3a was dissolved in ethanol to ˜100 mg/ml Injection: 5 ml per injection.

Work Up:

After separation, the fractions were dried off via rotary evaporator at bath temperature 40° C. to get the desired isomer Compound 3

which was the slower eluting enantiomer, retention time 3.76 min (other enantiomer—Compound 3b

retention time 2.76 min).

A solution of 95 mg of enantiomerically pure Compound 3 (330 mmol) was dissolved in 2 mL HOAc. To this was added 0.13 mL of a 10-15% NaOCl_((aq)) solution via syringe. After ca. 30 min another 0.25 mL of 10-15% NaOCl_((aq)) solution was added to the reaction mixture which was stirred ca. 30 min before the addition of water and CH₂Cl₂. The layers were separated, the CH₂C2 layer was rinsed with NaHSO_(3 (aq)), and NaHCO_(3 (aq)). The solution was dried (MgSO₄), concentrated and chromatographed with 0-50% EtOAc in hexane to yield 52 mg of Compound 6 as a white solid (49%).

¹H NMR (400 MHz, CDCl₃) δ 9.08 (s, 1H), 7.62-7.55 (m, 1H), 7.40 (dd, J=13.3, 2.1 Hz, 1H), 3.92-3.77 (m, 4H), 3.32-3.18 (m, 5H), 2.72 (dd, J=17.0, 6.9 Hz, 1H), 2.50 (d, J=17.0 Hz, 1H), 1.25 (d, J=7.4 Hz, 3H). ¹⁹F NMR (376 MHz, CDCl₃) δ −118.69. ¹³C NMR (101 MHz, CDCl₃) δ 166.50, 159.68 (d, J=249.9 Hz), 151.35 (d, J=2.9 Hz), 137.26 (d, J=13.4 Hz), 133.00 (d, J=7.3 Hz), 131.36 (d, J=8.9 Hz), 123.41 (d, J=2.6 Hz), 112.97 (d, J=24.0 Hz), 67.57, 51.08 (d, J=4.7 Hz), 33.72, 27.84, 16.23. Mass 326 (M+1).

Chiral SCF chromatography of the product showed no loss of enantiomeric purity:

Column: ChiralPak AS-H, 250×4.6 mm, 5 um,

Mobile Phase Modifier: 100% Methanol,

Gradient: 5 to 50% Methanol over 10 minutes,

Flow Rate: 4 mL/min,

Back Pressure: 100 bar,

Column Temperature: 40° C.

UV detection was from 200-400 nm.

Retention times of separated enantiomers: 6.11 ((R)) and 8.82 ((S)) min.

Analytical Separation of the Enantiomers—Compound 6a.

Instrument: Waters Acquity UPC2

Column: ChiralPak AS-H, 250×4.6 mm I.D.

Mobile phase: A for C02 and B for MeOH

Gradient: 3 to 50B 5 min, 10 min run

Flow rate: 1.5 mL/min

Back pressure: 100 bar

Column temperature: 45° C.

Wavelength: 210 nm

Retention time of Compound 6

-7.23 min; retention time of inactive enantiomer Compound 6b

-6.28 min.

Compound 6 was tested in the HeLa cell viability assay and its EC50 was determined to be 1.1 nM.

Attachment to Resin

To a solution of 18 mg of DNMDP-2L (0.04 mmol) in 0.8 mL of CH₂Cl₂ was added 0.2 mL of trifluoroacetic acid (TFA) and the solution was stirred 2 h before concentration and dissolution in 0.5 mL DMSO. To this was added 10 uL of Et3N (0.07 mmol) and 12 mg of N,N′-disuccinimidyl carbonate (DSC) (0.05 mmol) and the solution was stirred overnight. LC analysis indicated the reaction was not complete, another 25 mg of N,N′-disuccinimidyl carbonate (0.1 mmol) was added. LC analysis after 2 hours showed ca. 5:1 ratio of DSC product:amine. A 1 mL sample of Affi-Gel 102 resin was rinsed five times with DMSO with a centrifuge, then suspended in 0.5 mL DMSO. To the resin was added 30 uL of the DSC product solution and 25 uL Et3N and the mixture was swirled. After 2 days, LC analysis of the DMSO solution showed complete disappearance of the DCS adduct; the underivatized amine was still present. The DMSO was removed by centrifuge and decanted and the resin was rinsed several times with DMSO and stored in PBS buffer.

Bioactives Screen to Rescue DNMDP Induced Cytotoxicity

1000 HeLa cells were plated in a 384-well plate in 40 g of DMEM supplemented with 10% Fetal Bovine Serum and 1% Pen/Strep. 24 hours after plating, a compound-library of 1600 bioactive molecules (Pharmacon) was added at a concentration of 2 μM. In parallel to bioactive compound incubation, DNMDP was added to a final concentration of 30 nM and incubated for 48 hours. Cell viability was assessed as described in Compound library screening in NCI-H1734 and A549 cell lines.

Linker-Affinity Purification of Molecular Target of DNMDP and Immunoblotting

HeLa cells were washed with ice-cold PBS before lysed with NP-40 lysis buffer (150 mM NaCl, 10% glycerol, 50 mM Tris-Cl pH 8.0, 50 mM MgCl₂, 1% NP-40) supplemented with EDTA-free protease inhibitors (Roche) and phosphatase inhibitor mixtures I and II (Calbiochem). Cell lysates were incubated on ice for at least 2 minutes and subsequently centrifuged for 10 minutes at 4° C. at 15,700×g after which the supernatant was quantified using BCA protein assay kit (Pierce). 200 g total HeLa cell lysate was incubated with 3 l Affi-Gel 102 resin (BioRad) coupled to affinity linker DNMDP-2L in a total volume of 400 g for four hours. Prior to incubation, indicated compounds were added to affinity purifications at a final concentration of 1 μM. Samples were washed three times with lysis buffer containing corresponding compound concentrations of 1 μM. Proteins bound to Affi-Gel 102 resin were reduced, denatured, and separated using Tris-Glycine gels (Novex) and transferred to nitrocellulose membranes using the iBlot transfer system (Novex). Membranes were incubated overnight at 4° C. with primary antibodies against PDE3A (1:1000, Bethyl). Incubation with secondary antibodies (1:20,000, LI-COR Biosciences) for two hours at room temperature and subsequent detection (Odyssey Imaging System, LI-COR Biosciences) were performed according to manufacturer's recommendations.

PARP-Cleavage Immunoblotting

HeLa cells were treated with indicated concentration of DNMDP and staurosporine for 36 hours. HeLa cells were lysed and processed as described in Linker-affinity purification of molecular target of DNMDP and immunoblotting. Membranes were incubated with an antibody against PARP (1:1000, Cell Signaling #9532) and actin and subsequently imaged as described in Linker-affinity purification of molecular target of DNMDP and immunoblotting.

Targeting PDE3A Locus Using CRISPR

CRISPR target sites were identified using the MIT CRISPR Design Tool (online MIT CRISPR design portal). For cloning of sgRNAs, forward and reverse oligonucleotides (oligos) were annealed, phosphorylated and ligated into BsmBI-digested pXPR_BRD001. Oligo sequences are as follows:

sgRNA Forward oligo Reverse oligo PDE3A_sg1 CACCGTTTTCACTGAG AAACTCACTTCGCTCA CGAAGTGA GTGAAAAC (SEQ ID NO.: 7) (SEQ ID NO.: 8) PDE3A_sg2 CACCGAGACAAGCTTG AAACTTGGAATAGCAA CTATTCCAA GCTTGTCTC (SEQ ID NO.: 9) (SEQ ID NO.: 10) PDE3A_sg3 CACCGGCACTCTGAGT AAACTAACTTACACTC GTAAGTTA AGAGTGCC (SEQ ID NO.: 11) (SEQ ID NO.: 12) To produce lentivirus, 293T cells were co-transfected with pXPR_BRD001, psPAX2 and pMD2.G using calcium phosphate. Infected HeLa cells were selected with 2 ug/ml of puromycin. Reduction of PDE3A Expression Using siRNA

HeLa cells were plated in 96-well plates and transfected after 24 hours with PDE3A and Non-Targeting siRNA smartpools (On Target Plus, Thermo Scientific) according to the manufacturers recommendations. HeLa cell lysate was obtained 24 hours and 72 hours after transfection and immunoblotted for PDE3A and Actin (1:20,000, Cell Signaling) as described in Linker-affinity purification of molecular target of DNMDP and immunoblotting. HeLa cells were treated for 48 hours with indicated concentrations of Compound 3. Cell viability was assessed as described in Compound library screening in NCI-H1734 and A549 cell lines.

Measuring Cellular cAMP Concentrations in HeLa Cells

5000 HeLa cells were plated in 96-well plates. 24 hours after plating, HeLa cells were incubated for one hour with indicated compounds at indicated concentrations. cAMP levels were determined with the CAMP-GLO™ assay (Promega) according to the manufacturers recommendations. Cellular concentrations of cAMP were determined by normalizing to a standard curve generated according to the manufacturers recommendations.

Extended Proteomics Methods for PDE3A-Protein Interaction Studies Immunoprecipitation of PDE3A in HeLa Cells

HeLa cells were treated for four hours prior to lysis with 1 μM of indicated compounds: DMSO, DNMDP and trequinsin. HeLa cells were lysed with ModRipa lysis buffer (1% NP-40: 50 mM Tris-HCl, pH 7.8, 150 mM NaCl, 0.1% sodium deoxycholate, 1 mM EDTA) supplemented with protease and phosphatase inhibitors as described in Linker-affinity purification of molecular target of DNMDP and immunoblotting, and indicated compounds as described above to a final concentration of 10 μM. 13 mg of HeLa total cell lysate was incubated with 0.5% PDE3A antibody (Bethyl) and incubated overnight. Blocking peptide (Bethyl) against the PDE3A antibody was added simultaneously with the PDE3A antibody in the corresponding condition. Total cell lysate and antibody mixture was then incubated with 10 g Protein A Plus Agarose (Fisher Scientific) for 30 minutes at 4° C. Protein A Plus Agarose was then washed two times with lysis buffer containing indicated compounds at a concentration of 1 μM. Finally, Protein A Plus Agarose was washed once with lysis buffer containing no NP-40 and indicated compounds at a concentration of 1 μM.

On-Bead Digest

The beads from immunopurification were washed once with IP lysis buffer, then three times with PBS, the three different lysates of each replicate were resuspended in 90 uL digestion buffer (2M Urea, 50 mM Tris HCl), 2 ug of sequencing grade trypsin added, 1 hour shaking at 700 rpm. The supernatant was removed and placed in a fresh tube. The beads were then washed twice with 50 uL digestion buffer and combined with the supernatant. The combined supernatants were reduced (2 uL 500 mM DTT, 30 minutes, room temperature), alkylated (4 uL 500 mM IAA, 45 minutes, dark) and a longer overnight digestion performed: 2 ug (4 uL) trypsin, shake overnight. The samples were then quenched with 20 uL 10% folic acid (FA) and desalted on 10 mg SEP-PAK® columns.

iTRAQ Labeling of Peptides and Strong Cation Exchange (Scx) Fractionation

Desalted peptides were labeled with isobaric tags for relative and absolute quantification (iTRAQ)-reagents according to the manufacturer's instructions (AB Sciex, Foster City, Calif.). Peptides were dissolved in 30 μl of 0.5 M TEAB pH 8.5 solution and labeling reagent was added in 70 ul of ethanol. After 1 hour incubation the reaction was stopped with 50 mM Tris/HCl pH 7.5. Differentially labeled peptides were mixed and subsequently desalted on 10 mg SEP-PAK® columns.

iTRAQ labeling 114 115 116 117 Rep1 Blocking peptide No addition DNMDP trequinsin Rep2 Blocking peptide No addition DNMDP trequinsin SCX fractionation of the differentially labelled and combined peptides was done as described in Rappsilber et al. (Rappsilber et al., Nat Protoc 2, 1896-1906, 2007), with 6 pH steps (buffers—all contain 25% acetonitrile) as below:

1: ammonium acetate 50 mM pH 4.5,

2: ammonium acetate 50 mM pH 5.5,

3: ammonium acetate 50 mM pH 6.5,

4: ammonium bicarbonate 50 mM pH 8,

5: ammonium hydroxide 0.1% pH 9,

6: ammonium hydroxide 0.1% pH 11.

Empore SCX disk used to make stop-and-go-extraction-tips (StageTips) as described in the paper.

MS Analysis

Reconstituted peptides were separated on an online nanoflow EASY-NLC™ 1000 UHPLC system (Thermo Fisher Scientific) and analyzed on a benchtop Orbitrap Q EXACTIVET mass spectrometer (Thermo Fisher Scientific). The peptide samples were injected onto a capillary column (PICOFRIT® with 10 m tip opening/75 μm diameter, New Objective, PF360-75-10-N-5) packed in-house with 20 cm C18 silica material (1.9 m REPROSIL-PUR® C18-AQ medium, Dr. Maisch GmbH, r119.aq). The UHPLC setup was connected with a custom-fit microadapting tee (360 μm, IDEX Health & Science, UH-753), and capillary columns were heated to 50° C. in column heater sleeves (Phoenix-ST) to reduce backpressure during UHPLC separation. Injected peptides were separated at a flow rate of 200 nL/min with a linear 80 min gradient from 100% solvent A (3% acetonitrile, 0.1% formic acid) to 30% solvent B (90% acetonitrile, 0.1% formic acid), followed by a linear 6 min gradient from 30% solvent B to 90% solvent B. Each sample was run for 120 minutes, including sample loading and column equilibration times. The Q EXACTIVE™ instrument was operated in the data-dependent mode acquiring high-energy collisional dissociation (HCD) MS/MS scans (R=17,500) after each MS1 scan (R=70,000) on the 12 top most abundant ions using an MS1 ion target of 3×106 ions and an MS2 target of 5×104 ions. The maximum ion time utilized for the MS/MS scans was 120 ms; the HCD-normalized collision energy was set to 27; the dynamic exclusion time was set to 20s, and the peptide match and isotope exclusion functions were enabled.

Quantification and Identification of Peptides and Proteins

All mass spectra were processed using the Spectrum Mill software package v4.1 beta (Agilent Technologies) which includes modules developed by Applicants for isobaric tags for relative and absolute quantification (iTRAQ)-based quantification. Precursor ion quantification was done using extracted ion chromatograms (XIC's) for each precursor ion. The peak area for the XIC of each precursor ion subjected to MS/MS was calculated automatically by the Spectrum Mill software in the intervening high-resolution MS1 scans of the liquid chromatography (LC)-MS/MS runs using narrow windows around each individual member of the isotope cluster. Peak widths in both the time and m/z domains were dynamically determined based on MS scan resolution, precursor charge and m/z, subject to quality metrics on the relative distribution of the peaks in the isotope cluster vs theoretical. Similar MS/MS spectra acquired on the same precursor m/z in the same dissociation mode within +/−60 seconds were merged. MS/MS spectra with precursor charge >7 and poor quality MS/MS spectra, which failed the quality filter by not having a sequence tag length >1 (i.e., minimum of 3 masses separated by the in-chain mass of an amino acid) were excluded from searching.

For peptide identification MS/MS spectra were searched against human Universal Protein Resource (Uniprot) database to which a set of common laboratory contaminant proteins was appended. Search parameters included: ESI-Q EXACTIVE™-HCD scoring parameters, trypsin enzyme specificity with a maximum of two missed cleavages, 40% minimum matched peak intensity, +/−20 ppm precursor mass tolerance, +/−20 ppm product mass tolerance, and carbamidomethylation of cysteines and iTRAQ labeling of lysines and peptide n-termini as fixed modifications. Allowed variable modifications were oxidation of methionine, N-terminal acetylation, Pyroglutamic acid (N-termQ), Deamidated (N), Pyro Carbamidomethyl Cys (N-termC), with a precursor MH+ shift range of −18 to 64 Da. Identities interpreted for individual spectra were automatically designated as valid by optimizing score and delta rank1-rank2 score thresholds separately for each precursor charge state in each liquid chromatography (LC)-MS/MS while allowing a maximum target-decoy-based false-discovery rate (FDR) of 1.0% at the spectrum level.

In calculating scores at the protein level and reporting the identified proteins, redundancy is addressed in the following manner: the protein score is the sum of the scores of distinct peptides. A distinct peptide is the single highest scoring instance of a peptide detected through an MS/MS spectrum. MS/MS spectra for a particular peptide may have been recorded multiple times, (i.e. as different precursor charge states, isolated from adjacent SCX fractions, modified by oxidation of Met) but are still counted as a single distinct peptide. When a peptide sequence >8 residues long is contained in multiple protein entries in the sequence database, the proteins are grouped together and the highest scoring one and its accession number are reported. In some cases when the protein sequences are grouped in this manner there are distinct peptides which uniquely represent a lower scoring member of the group (isoforms or family members). Each of these instances spawns a subgroup and multiple subgroups are reported and counted towards the total number of proteins. iTRAQ ratios were obtained from the protein-comparisons export table in Spectrum Mill. To obtain iTRAQ protein ratios the median was calculated over all distinct peptides assigned to a protein subgroup in each replicate. To assign interacting proteins the Limma package in the R environment was used to calculate moderated t-test p, as described previously and added Blandt-Altman testing to filter out proteins for which the CI for reproducibility was below 95% (Udeshi et al., Mol Cell Proteomics 11, 148-159, 2012).

Validation of DNMDP-Induced PDE3A Protein Interactions Using Immunoprecipitation and Immunoblotting

HeLa cells were transfected with ORF overexpression constructs expressing V5-tagged SIRT7, V5-tagged SLFN12, or V5-tagged GFP. ORF expression constructs were obtained from the TRC (clone IDs: TRCN0000468231, TRCN0000476272, ccsbBroad304_99997). At 72 hours post transfection, cells were treated with 1 μM DNMDP or trequinsin for 4 hours followed by lysis using the ModRipa lysis buffer and immunoprecipitation of PDE3A. For each condition, 2 mg total protein lysate was incubated with 1 g of anti-PDE3A antibody at 4° C. overnight, after which 7.5 μl each of Protein A- and Protein G-Dynabeads (Life Technologies 10001D and 10003D) were added and incubated for another 1 hour. Beads were washed and bound proteins were eluted with 30 j of LDS PAGE gel loading buffer. Input (60 g total protein lysate) and IP products were resolved on 4-12% Tris-Glycine PAGE gels and immunoblotted with an anti-V5 antibody (Life Technologies R96205, 1:5000), the Bethyl anti-PDE3A antibody (1:1000), and secondary antibodies from LiCOR Biosciences (Cat. #926-32210 and 926068021, each at 1:10,000). Blots were washed and imaged using a LiCOR Odyssey infrared imager.

Knockdown of SLFN12 Expression Using shRNA and Testing for Drug Sensitivity

Constructs expressing shRNAs targeting SLFN12, or the control vector, were packaged into lentiviruses and delivered into HeLa cells by viral transduction. Three SLFN12-targeting shRNAs were used, all of which were obtained from the TRC (CloneIDs: TRCN0000152141 and TRCN0000153520). Infected cells were selected using 1 g/ml puromycin for 3 days and then grown in non-selective media for 3 more days. Cells were then plated into 384-well assay plates and tested for drug sensitivity as described above. Knockdown of SLFN12 was validated by qPCR. Total RNA was extracted using kit reagents (RNeasy Mini Kit (Qiagen #74104) and QlAschredder (Qiagen #79656)). cDNA was generated using kit reagents (SuperScript III First-Strand Synthesis System (Life Technologies #18080-051)). qPCR was performed for GAPDH and SLFN12 (Life Technologies Hs00430118_ml) according to the manufacturer's recommendations. SLFN12 expression was normalized to corresponding samples GAPDH ct-values.

Other Embodiments

From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

INCORPORATION BY REFERENCE

All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference. 

1-4. (canceled)
 5. A method of identifying a subject having a cancer that is resistant to PDE3A modulation, the method comprising detecting a decrease in the level of a CREB3L1 polypeptide or polynucleotide level in a biological sample of the subject relative to a reference, thereby identifying said subject as having a cancer resistant to PDE3A modulation.
 6. The method of claim 5, further comprising detecting a decrease in the level of SLFN12. 7-12. (canceled)
 13. A kit for identifying a subject having cancer that is resistant to PDE3A modulation, the kit comprising a capture reagent that binds CREB3L1 polypeptide or polynucleotide.
 14. The kit of claim 13, further comprising a capture reagent that binds SLFN12 polypeptide or polynucleotide.
 15. A compound having the structure:

or a pharmaceutically acceptable salt, or prodrug thereof.
 16. A pharmaceutical composition comprising one or more pharmaceutically acceptable carriers or excipients and a compound having the structure:

or a pharmaceutically acceptable salt, or prodrug thereof. 17-20. (canceled)
 21. A kit for decreasing cancer cell proliferation in a subject pre-selected as responsive to a PDE3A modulator, the kit comprising a compound having the structure:

or a pharmaceutically acceptable salt, or prodrug thereof. 22-23. (canceled)
 24. A method of preparing compound 6 according to claim 15, said method comprising the step of reacting the racemate Compound 3a

with NaOCl in acetic acid at a temperature range form 10-15° C. (including 10° and 15°) to obtain the racemate Compound 6a

and subsequently performing a separation of enantiomers of Compound 6a to obtain Compound 6


25. A method according to claim 24 further comprising optionally in a preceding step a separation of enantiomers of compound 3a to obtain Compound 3

and the reaction step with NaOCl in acetic acid at a temperature range form 10-15° C. (including 10° and 15°) is performed with the enantiomer Compound 3 and optionally further comprising a subsequent separation of enantiomers.
 26. A method according to claim 24 whereby the enantiomer Compound 3

is reacted to obtain enantiomer Compound 6


27. Use of compound 3

for the preparation of compound
 6. 